Purified mammalian CTLA-8 antigens and related reagents

ABSTRACT

CTLA-8 antigen from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding said antigen. Methods of using said reagents and diagnostic kits are also provided.

[0001] The present application is a continuation-in-part of copendingU.S. Ser. No. 08/250,846, filed May 27, 1994, which is acontinuation-in-part of then copending patent application U.S. Ser. No.08/177,747, filed Jan. 5, 1994, which is a continuation-in-part of thencopending patent application U.S. Ser. No. 08/077,203, filed Jun. 14,1993, each of which is incorporated herein by reference. Alsoincorporated by reference is co-pending PCT/US95/00001, filed Jan. 3,1995.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions related to proteinswhich function in controlling physiology, development, anddifferentiation of mammalian cells, e.g., cells of a mammalian immunesystem. In particular, it provides proteins and mimetics which regulatecellular physiology, development, differentiation, or function ofvarious cell types, including hematopoietic cells.

BACKGROUND OF THE INVENTION

[0003] The immune system of vertebrates consists of a number of organsand several different cell types. Two major cell types include themyeloid and lymphoid lineages. Among the lymphoid cell lineage are Bcells, which were originally characterized as differentiating in fetalliver or adult bone marrow, and T cells, which were originallycharacterized as differentiating in the thymus. See, e.g., Paul (ed.)(1993) Fundamental Immunology (3d ed.) Raven Press, New York.

[0004] In many aspects of the development of an immune response orcellular differentiation, soluble proteins known as cytokines play acritical role in regulating cellular interactions. These cytokinesapparently mediate cellular activities in many ways. They have beenshown, in many cases, to modulate proliferation, growth, anddifferentiation of hematopoietic stem cells into the vast number ofprogenitors composing the lineages responsible for an immune response.

[0005] However, the cellular molecules which are expressed by differentdevelopmental stages of cells in these maturation pathways are stillincompletely identified. Moreover, the roles and mechanisms of action ofsignaling molecules which induce, sustain, or modulate the variousphysiological, developmental, or proliferative states of these cells ispoorly understood. Clearly, the immune system and its response tovarious stresses had relevance to medicine, e.g., infectious diseases,cancer related responses and treatment, allergic and transplantationrejection responses. See, e.g., Thorn, et al. Harrison's Principles ofInternal Medicine McGraw/Hill, New York.

[0006] Medical science relies, in large degree, to appropriaterecruitment or suppression of the immune system in effecting cures forinsufficient or improper physiological responses to environmentalfactors. However, the lack of understanding of how the immune system isregulated or differentiates has blocked the ability to advantageouslymodulate the normal defensive mechanisms to biological challenges.Medical conditions characterized by abnormal or inappropriate regulationof the development or physiology of relevant cells thus remainunmanageable. The discovery and characterization of specific cytokineswill contribute to the development of therapies for a broad range ofdegenerative or other conditions which affect the immune system,hematopoietic cells, as well as other cell types. The present inventionprovides solutions to some of these and many other problems.

SUMMARY OF THE INVENTION

[0007] The present invention is based, in part, upon the discovery of acDNA clone encoding a cytokine-like protein. This protein has beendesignated CTLA-8. The invention embraces isolated genes encoding theproteins of the invention, variants of the encoded protein, e.g.,mutations (muteins) of the natural sequence, species and allelicvariants, fusion proteins, chemical mimetics, antibodies, and otherstructural or functional analogs. Various uses of these differentnucleic acid or protein compositions are also provided.

[0008] The present invention embraces isolated genes encoding theproteins of the invention, variants of the encoded protein, e.g.,mutations (muteins) of the natural sequence, species and allelicvariants, fusion proteins, chemical mimetics, antibodies, and otherstructural or functional analogs. Various uses of these differentnucleic acid or protein compositions are also provided.

[0009] The present invention provides a nucleic acid with at least 95%identity to one encoding a mammalian CTLA-8 protein or fragment thereof.The encoding nucleic acid can comprise a sequence of SEQ ID NO: 1, 3, 5,7, or 9.

[0010] The present invention also provides a substantially puremammalian CTLA-8 protein or peptide thereof. The protein or peptide cancomprise at least one polypeptide segment of SEQ ID NO: 2, 4, 6, 8, or10; exhibit a post-translational modification pattern distinct from anatural mammalian CTLA-8 protein; or induce a cell to secrete aninflammatory mediator, e.g., IL-6, IL-8, and/or PGE2. A furtherembodiment is a composition comprising such a protein and apharmaceutically acceptable carrier.

[0011] The invention includes an antibody which specifically binds to aprimate CTLA-8 protein or peptide thereof; the antibody is raisedagainst a protein sequence of SEQ ID NO: 2, 4, 6, 8 or 10; the antibodyis a monoclonal antibody; the antibody blocks the CTLA-8 inducedsecretion of an inflammatory mediator, e.g., IL-6, IL-8, and/or PGE2; orthe antibody is labeled.

[0012] The invention also embraces a kit comprising a substantially purenucleic acid at least 95% identical to one encoding a mammalian CTLA-8protein or peptide; a substantially. pure mammalian CTLA-8 protein orfragment, e.g., as a positive control; or an antibody or receptor whichspecifically binds a mammalian CTLA-8 protein.

[0013] The availability of these reagents also provides methods ofmodulating physiology or development of a cell comprising contactingsaid cell with an agonist or antagonist of a CTLA-8 protein. The methodof modulation encompasses regulating CTLA-8 induced secretion of aninflammatory mediator, e.g., IL-6, IL-8, and/or PGE2, by contacting thecell or tissue with an antibody which specifically binds mammalianCTLA-8 or a substantially pure mammalian CTLA-8 protein. Preferably, thecell can be a synovial cell, epithelial cell, endothelial cell,fibroblast cell, or a carcinoma cell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OUTLINE

[0014] I. General

[0015] II. Nucleic Acids

[0016] A. natural isolates; methods

[0017] B. synthetic genes

[0018] C. methods to isolate

[0019] III. Purified CTLA-8 protein

[0020] A. physical properties

[0021] B. biological properties

[0022] IV. Making CTLA-8 protein; Mimetics

[0023] A. recombinant methods

[0024] B. synthetic methods

[0025] C. natural purification

[0026] V. Physical Variants

[0027] A. sequence variants, fragments

[0028] B. post-translational variants

[0029] 1. glycosylation

[0030] 2. others

[0031] VI. Functional Variants

[0032] A. analogs; fragments

[0033] 1. agonists

[0034] 2. antagonists

[0035] B. mimetics

[0036] 1. protein

[0037] 2. chemicals

[0038] C. species variants

[0039] VII. Antibodies

[0040] A. polyclonal

[0041] B. monoclonal

[0042] C. fragments, binding compositions

[0043] VIII. Uses

[0044] A. diagnostic

[0045] B. therapeutic

[0046] IX. Kits

[0047] A. nucleic acid reagents

[0048] B. protein reagents

[0049] C. antibody reagents

[0050] I. General

[0051] The present invention provides DNA sequence encoding variousmammalian proteins which exhibit properties characteristic offunctionally significant T cell expressed molecules. The cDNA sequenceexhibits various features which are characteristic of mRNAs encodingcytokines, growth factors, and oncogenes. A murine gene originallythought to be from a mouse, but now recognized as rat as describedherein contains an open reading frame encoding a putative 150 amino acidprotein. This protein is 57% homologous to a putative protein encoded bya viral genome, the herpesvirus Saimiri ORF13. The message was isolatedusing a subtraction hybridization method applied to T cells.

[0052] These proteins are designated CTLA-8 proteins. The naturalproteins should be capable of mediating various physiological responseswhich would lead to biological or physiological responses in targetcells. Initial studies had localized the message encoding this proteinto various cell lines of hematopoietic cells. Genes encoding the antigenhave been mapped to mouse chromosome 1A and human chromosome 2q31.Murine CTLA-8 was originally cloned by Rouvier, et al. (1993) J.Immunol. 150:5445-5456. Similar sequences for proteins in othermammalian species should also be available.

[0053] Purified CTLA-8, when cultured with synoviocytes, is able toinduce the secretion of IL-6 from these cells. This induction isreversed upon the addition of a neutralizing antibody raised againsthuman CTLA-8-8. Endothelial, epithelial, fibroblast and carcinoma cellsalso exhibit responses to treatment with CTLA-8. This data suggests thatCTLA-8 may be implicated in inflammatory fibrosis, e.g., psoriasis,sclerodermia, lung fibrosis, or cirrhosis. CTLA-8 may also causeproliferation of carcinomas or other cancer cells inasmuch as IL-6 oftenacts as a growth factor for such cells.

[0054] The descriptions below are directed, for exemplary purposes, to amurine or human CTLA-8 protein, but are likewise applicable to relatedembodiments from other species.

[0055] II. Nucleic Acids

[0056] Table 1 discloses the nucleotide and amino acid sequences of amurine CTLA-8 protein. The described nucleotide sequences and therelated reagents are useful in constructing a DNA clone useful forexpressing CTLA-8 protein, or, e.g., isolating a homologous gene fromanother natural source. Typically, the sequences will be useful inisolating other genes, e.g., allelic variants, from mouse, and similarprocedures will be applied to isolate genes from other species, e.g.,warm blooded animals, such as birds and mammals. Cross hybridizationwill allow isolation of genes from other species. A number of differentapproaches should be available to successfully isolate a suitablenucleic acid clone from other sources. TABLE 1 Nucleotide sequenceencoding a murine CTLA-8 protein and predicted amino acid sequence. Alsocan use complementary nucleic acid sequences for many purposes.Submitted to GenBank/EMBL under accession number L13839. 1 GAATTCCATCCATGTGCCTG ATGCTGTTGC TGCTACTGAA CCTGGAGGCT ACAGTGAAGG 61 CAGCGGTACTCATCCCTCAA AGTTCAGTGT GTCCAAACGC CGAGGCCAAT AACTTTCTCC 121 AGAACGTGAAGGTCAACCTG AAAGTCATCA ACTCCCTTAG CTCAAAAGCG AGCTCCAGAA 181 GGCCCTCAGACTACCTCAAC CGTTCCACTT CACCCTGGAC TCTGAGCCGC AATGACGACC 241 CTGATAGATATCCTTCTGTG ATCTGGGAGG CACAGTGCCG CCACCAGCGC TGTGTCAACG 301 CTGAGGGGAAGTTGGACCAC CACATGAATT CTGTTCTCAT CCAGCAAGAG ATCCTGGTCC 361 TGAAGAGGGAGCCTGAGAAG TGCCCCTTCA CTTTCCGGGT GGAGAAGATG CTGGTGGGCG 421 TGGGCTGCACCTGCGTTTCC TCTATTGTCC GCCATGCGTC CTAAACAGAG ACCTGAGGCT 481 AGCCCCTAAGAAACCCCTGC GTTTCTCTGC AAACTTCCTT GTCTTITTAA AACAGTTCAC 541 AGTTGAATCTCAGCAAGTGA TATGGATTTA AAGGCGGGGT TAGAATTGTC TGCCTTCCAC 601 CCTGAAAAGAAGGCGCAGAG GGGATATAAA TTGCTTCTTG TTTTTCTGTG GGCTTTAAAT 661 TATTTATGTATTTACTCTAT CCCGAGATAA CTTTGAGGCA TAAGTTATTT TAATGAATTA 721 TCTACATTATTATTATGTTT CTTAATGCAG AAGACAAAAT TCAAGACTAA GAAATTTTAT 781 TATTTAAAAGGTAAAACCTA TATTTATATG AGCTATTTAT GGGTCTATTT ATTTTTCTTC 841 AGTGCTAAGATCATGATTAT CAGATCTACC TAAGGAAGTC CTAAATAATA TTAAATATTA 901 ATTGAAATTTCAGTTTTACT ATTTGCTTAT TTAAGGTTCC CTCCTCTGAA TGGTGTGAAA 961 TCAAACCTCGTTTTATGTTT TTAAATTATT GAGGCTTCGA AAAATTGGGC AATTTAGCTT 1021 CCTACTGTGTGTTTAAAAAC CTTGTAACAA TATCACTATA ATAAATTTTT GGAAGAAAAT predicted aminoacid sequence (150 amino acids). Mature polypeptide probably starts atabout amino acid 13 (Ala). MCLML LLLLN LEATV KAAVL IPQSS VCPNA EANNFLQNVK VNLKV INSLS SKASS RRPSD YLNRS TSPWT LSRNE DPDRY PSVIW EAQCR HQRCVNAEGK LDHHM NSVLI QQEIL VLKRE PEKCP FTFRV EKMLV GVGCT CVSSI VRHAS

[0057] The purified protein or defined peptides are useful forgenerating antibodies by standard methods, as described above. Syntheticpeptides or purified protein can be presented to an immune system togenerate a specific binding composition, e.g., monoclonal or polyclonalantibodies. See, e.g., Coligan (1991) Current Protocols in ImmunologyWiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory ManualCold Spring Harbor Press.

[0058] For example, the specific binding composition could be used forscreening of an expression library made from a cell line which expressesa CTLA-8 protein. The screening can be standard staining of surfaceexpressed protein, or by panning. Screening of intracellular expressioncan also be performed by various staining or immunofluorescenceprocedures. The binding compositions could be used to affinity purify orsort out cells expressing the protein.

[0059] This invention contemplates use of isolated DNA or fragments toencode a biologically active corresponding CTLA-8 protein orpolypeptide. In addition, this invention covers isolated or recombinantDNA which encodes a biologically active protein or polypeptide and whichis capable of hybridizing under appropriate conditions with the DNAsequences described herein. Said biologically active protein orpolypeptide can be an intact antigen, or fragment, and have an aminoacid sequence as disclosed in Table 1. Further, this invention coversthe use of isolated or recombinant DNA, or fragments thereof, whichencode proteins which are homologous to a CTLA-8 protein or which wereisolated using cDNA encoding a CTLA-8 protein as a probe. The isolatedDNA can have the respective regulatory sequences in the 5′ and 3′flanks, e.g., promoters, enhancers, poly-A addition signals, and others.In particular, the murine CTLA-8 gene has significant homology, about60%, to the putative protein encoded by the open reading frame ORF13, ofherpesvirus Saimiri (Table 2); to a human CTLA-8 counterpart (Table 3),about 60%; and to a mouse CTLA-8 counterpart (Table 4), about 80%. TABLE2 Nucleotide sequence of the related herpesvirus Saimiri open readingframe ORF13 and predicted amino acid sequence of encoded protein, seeGenBank/EMBL accession number M60286. herpesvirus ACCTTCATGC AAATACATCTTATCTTACCA GATTCTCGCC TCATTTGCAA 50 ACATGCCTCA TCTTTTGAGA AGAAACGCAATTCGAACTTC TTCTAATGCT 100 CCTGAACAGC AGCCTGTGCT GCAGCCTGAG CTTGATGCTATTGAAGAGCT 150 AGAATAAGAG CTATTTTTTG ACGATGGGTG CTGCCTTTCT GTTCAAGAAA200 TCTGCTTAAT TGTTCTTGGA TTCTTATTGT TTCTGCTAGC TGTAATTGTT 250TTTTATAACT ATACAGACAC AGATCAATTT GTGAAGCTGA CACATCTTAT 300 GAGCCACAAAAATTCTATCA AAGGACCTTT AAGGACCTTT GGTATGTACT 350 CATAATTTTA TTTTTTTATTTCTAAAACAA TCTTAGTATA TATAATTAAT 400 ACAAATTTTA GAAAATACTA TAATAAATATTGAAAGCTGT ATTTACATTG 450 TAAACTATAT ATAGGCAATG TAAAGTCATT CTAACTTTAGGTTTGCTTTA 500 CCTGTTACAG AAACTTCACC TGTGTGTCAA GAGCTGCAAA CATGGCTTTA550 GACTTAAGAA ATCTTAAACA CCTGACTGCT AACTTCAGTT TTAGAATAAT 600GATATGGATT ATGCTATGTT TGGCTCTACC TACTGATAGT AAACCTATTT 650 CAACAACTGAAGCTCCAATA CTAAACATAA CACAATCTCC AAGTTTGAAC 700 ATCTCATCAC CTTCTACTTTAGAACCTTCA GAGCCTCTTA AAAACTGTAC 750 AACATTCTTA GACTTACTTT GGCAGCGGCTGGGCGAGAAC GCTTCTATAA 800 AGGACTTGAT GTTAACATTA CAACGAGAAG AAGTCCACGGAAGAATGACT 850 ACACTTCCTT CACCTAGACC AAGCAGTAAA GTTGAAGAAC AACAGTTACA900 AAGACCTAGA AACTTACTGC CTACTGCTGT CGGGCCACCT CATGTCAAAT 950ATAGACTATA TAATCGCTTA TGGGAAGCTC CTAAAGGAGC TGATGTTAAT 1000 GGTAAACCTATACAATTTGA TGACCCTCCT CTTCCTTATA CAGGGGCATA 1050 TAATGATGAT GGTGTTTTAATGGTTAATAT TAATGGAAAA CATGTGAGGT 1100 TTGATAGCTT GTCTTATTGG GAAAGAATTAAAAGATCTGG TACCCCATGG 1150 TGTATAAAGA CACCAAGTGA AAAAGCAGCA ATATTGAAGCAGCTTTTAAA 1200 AGCTGAAAAA AAATGTAGGA CTACTTCTAA ACGTATCACT GAGTTAGAAG1250 AGCAGATTAA AGAACTAGAA AAAACTAGTA CATCTCCATA GATTACTGTT 1300AGAATGTGTT TATCATACTA AAATAAATGC TTTATGTATT GCAATATTAC 1350 TTGTTTGCTATGACTTTGGT ATATGAAATG CAAATCTTAA ATAAAAAGTT 1400 TTTCTCTAGT ATTGGCGTCACTGTATTTTA CTAGCAAAAA TATATAAATT 1450 GTTATGTAGC AACAAGTTTG TATCAATATAAAAACTCTAA AGTATATAAA 1500 CAAACATTCA ATTAGTGTAA ATCATAGCAA GCATATCTTTTCATACGTGT 1550 CTAGTTAATT TAAAGAATTA ATTATGACAT TTAGAATGAC TTCACTTGTG1600 TTACTTCTCC TGCTGAGCAT AGATTGTATA GTAAAGTCAG AAATAACAAG 1650CGCACAAACC CCAAGATGCT TAGCTGCTAA CAATAGCTTT CCACGGTCTG 1700 TGATGGTTACTTTGAGCATC CGTAACTGGA ATACTAGTTC TAAAAGGGCT 1750 TCAGACTACT ACAATAGATCTACGTCTCCT TGGACTCTCC ATCGCAATGA 1800 AGATCAAGAT AGATATCCCT CTGTGATTTCGGAAGCAAAG TGTCGCTACT 1850 TACGATGTGT TAATGCTGAT GGGAATGTAG ACTACCACATGAACTCAGTC 1900 CCTATCCAAC AAGAGATTCT AGTGGTGCGC AAAGGGCATC AACCCTGCCC1950 TAATTCATTT AGGCTAGAGA AGATGCTAGT GACTGTAGGC TGCACATGCG 2000TTACTCCCAT TGTTCACAAT GTAGACTAAA AGCTATCTAA ATTTTGAAAA 2050 TTAACATTTCACTAAAAAAC AAAAACTTGA TTTTTTTCTT TTAAATAAAA 2100 AAAGTTTAAT ATAAGTTCTGGCTTGTTTGG TTTTTGACTA ATCAATGTAG 2150 ATCACACTTG TGATCTTAGC TCTCGGGAAGCAATGTAAGA AAATATATTT 2200 AACTTAAGAG TTTTAGACTT GCTTGAGTTT TATGAGTAAAAAACAAAGAA 2250 TAAGCACAGC TTCTTGTATC TTCTTTTAAA AACTTTAAGT TATTTATGTA2300 TTTAATATAA TCTAATGTTT CTTAAACATG TTGAGTTTGA GGTCCACTAA 2350TACAACATTA TAATTTTTTC TGTTATAACA CTTTTGCAAG AAGAACTCAT 2400 TTTATAGAAAATGAGCAGTA TTCAAAAAAA ATGTTTGATA TGCTGTAATA 2450 TTGGAGAGGA AGAACTTTTACAAGCATGTG ATTGTCCTAG CAGAGTCCAT 2500 CATACATGCT TACAAAGTCA 2520 Tm17Peptide sequence MVIDG CKKYM RRTCG DVLDN LRGDC YQVLI EDCIP VLKMY AKEGREFDYV 50 INDLT AVPIS TSPEE DSTWD FLRLI LDLSM KVLKQ DGKTF TQGNC VNLTE 100ALSLY EEQLG RLYCP VEFSK EIVCV PSYLE LNVFY TVWKK AKP 143

[0060] TABLE 3 Nucleotide sequence of human CTLA-8 fragment andpredicted amino acid sequence of encoded protein. AGC/CGC AAT GAG GACCCT GAG AGA TAT CCC TCT GTG ATC TGG GAG GCA AAG TGC CGC CAC TTG CCC TGCATC AAC GCT GAT GGG AAC GTG GAC TAC CAC ATG AAC TCT GTC CCC ATC CAG CAAGAG ATC CTG GTC CTG CGC AGG GAG CCT CCA CAC TGC CCC AAC TCC TTC CGG CTGGAG AAG ATA CTG GTG TCC GTG GGC TCC ACC TCT GTC ACC CCC ATT GTC CAC CATGTG GCC TAA ser/arg asn glu asp pro glu arg tyr pro ser val ile trp gluala lys cys arg his leu gly cys ile asn ala asp gly asn val asp tyr hismet asn ser val pro ile gln gln glu ile leu val leu arg arg glu pro prohis cys pro asn ser phe arg leu glu lys ile leu val ser val gly cys thrcys val thr pro ile val his his val ala OCH this was used to isolate afull length clone from human; it corresponds to nucleotides 272-510:             GG CACAAACTCA TCCATCCCCA GTTGATTGGA AGAAACAACG 42 ATG ACTCCT GGG AAG ACC TCA TTG GTG TCA CTG CTA CTG CTG CTG 87 Met thr pro glylys thr ser leu val ser leu leu leu leu leu 15 AGC CTG GAG GCC ATA GTCAAG GCA GGA ATC ACA ATC CCA CGA AAT 132 ser leu glu ala ile val lys alagly ile thr ile pro arg asn 30 CCA GGA TCC CCA AAT TCT GAG GAC AAG AACTTC CCC CGG ACT GTG 177 pro gly cys pro asn ser glu asp lys asn phe proarg thr val 45 ATG GTC AAC CTG AAC ATC CAT AAC CGG AAT ACC AAT ACC AATCCC 222 met val asn leu asn ile his asn arg asn thr asn thr asn pro 60aaA AGG TCC TCA GAT TAC TAC AAC CGA TCC ACC TCA CCT TGG AAT 267 lys argser ser asp tyr tyr asn arg ser thr ser pro trp asn 75 CTC CAC CGC AATGAG GAC CCT GAG AGA TAT CCC TCT CTG ATC TGG 312 leu his arg asn glu asppro glu arg tyr pro ser val ile trp 90 GAG GCA AAG TGC CGC CAC TTG GGCTGC ATC AAC GCT GAT GGG AAC 357 glu ala lys cys arg his leu gly cys ileasn ala asp gly asn 105 GTG GAC TAC CAC ATG AAC TCT GTC CCC ATC CAG CAAGAG ATC CTG 402 val asp tyr his met asn ser val pro ile gln gln glu ileleu 120 GTC CTG CGC AGG GAG CCT CCA CAC TGC CCC AAC TCC TTC CGG CTG 447val leu arg arg glu pro pro his cys pro asn ser phe arg leu 135 GAG AAGATA CTG GTG TCC GTG GGC TGC ACC TGT GTC ACC CCG ATT 492 glu lys ile leuval ser val gly cys thr cys val thr pro ile 150 GTC CAC CAT GTG GCC TAA510 val his his val ala OCH 156

[0061] TABLE 4 Nucleotide sequence of mouse CTLA-8 fragment andpredicted amino acid sequence of encoded protein.gaggctcaagtgcacccagcaccagctgatcaggacgcgcaaacatgagtccagggagagcttcatctg 69tgtctctgatgctgttgctgctgctgagcctggcggctacagtgaaggcagcagcgatcatccctcaaa138gctcagcgtgtccaaacactgaggccaaggacttcctccagaatgtgaaggtcaacctcaaagtcttta207actcccTTGGCGCAAAAGTGAGCTCCAGAAGgCCCTCAGACTACCTCAACCGTTCCACGTCACCCTGGA276CTCTCCACCGCAATGAAGACCCTGATAGATATCCCTCTGTGATCTGGGAAGCTCAGTGCCGCCACCAGC345GCTGTGTCAATGCGGAGggaaagctggaccaccacatgaattctgttctcatccagcaagagatcctgg414tcctgaagagggagcctgagagctgccccttcactttcagggtcgagaagatgctggtgggTGTGGGCT483GCACCTGCGTGGCCTCGATTGTCCGCCAGGCAGCCTAAACAGAGACCCGCGGCTGACCCCTAAGAAACC552CCCACGTTTCTCAGCAAACTTACTTGCATTTTTAAAACAGTTCGTGCTATTGATTTTCAGCAAGGAATG621TGGATTCAGAGGCAGATTCAGAATTGTCTGCCCTCCACAATGAAAAGAAGGTGTAAAGGGGTCCCAAAC690TGCTTCgtgtttgtttttctgtggactttaaattatttgtgtatttacaatatcccaagataactttga759aggcgtaacttatttaatgaagtatctacattattattatgtttctttctgaagaagacaaaattcaag828actcagaaattttattatttaaaaggtaagcctatatttatatgagctatttatgaatctatttatttt897tcttcagtatttgaagtattaagaacatgattttCAGATCTACCTACGGAAGTCCTAAGTAAGATTAAA966TATTAATGGAAATTTCAGCTTTACTATTTGGTTGATTTAAGGTTCTCTCCTCTGAATGGGGTGAAAACC1035AAACTTAGTTTTATGTTTAATAACTTTTTAAATTATTGAAGATTCAAAAAATTGGATAATTTAGCTCCC1104 TACTCTGTTTTAAAAAAAAAAAAAAAAAAA 1134 Mouse CTLA-8 predicted aminoacid sequence. The mature polypeptide probably starts at a positionabout amino acid 19 (Leu) to amino acid 21 (Ala).METSerProGlyArgAlaSerSerValSerLeuMETLeuLeuLeuLeuLeuSerLeuAlaAlaThrValLys24AlaAlaAlaIleIleProGlnSerSerAlaCysProAsnThrGluAlaLysAspPheLeuGlnAsnValLys48ValAsnLeuLysValPheAsnSerLeuGlyAlaLysValSerSerArgArgProSerAspTyrLeuAsnArg72SerThrSerProTrpThrLeuHisArgAsnGluAspProAspArgTyrProSerValIleTrpGluAlaGln96CysArgHisGlnArgCysValAsnAlacluGlyLysLeuAspHisHisMETAsnSerValLeuIleGlnGln120GluIleLeuValLeuLysArgGluProGluSercysProPheThrPheArgValGlubysMETLeuValGly144 ValGlyCysThrCysValAlaSerIleValArgGlnAlaAla 158

[0062] An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA,or a mixed polymer, which is substantially separated from othercomponents which naturally accompany a native sequence, e.g., ribosomes,polymerases, and flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Alternatively, a purified species may be separated from host componentsfrom a recombinant expression system. The size of homology of such anucleic acid will typically be less than large vectors, e.g., less thantens of kB, typically less than several kB, and preferably in the 2-6 kBrange.

[0063] An isolated nucleic acid will generally be a homogeneouscomposition of molecules, but will, in some embodiments, contain minorheterogeneity. This heterogeneity is typically found at the polymer endsor portions not critical to a desired biological function or activity.

[0064] A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, for example, products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site. Alternatively, it is performed tojoin together nucleic acid segments of desired functions to generate asingle genetic entity comprising a desired combination of functions notfound in the commonly available natural forms. Restriction enzymerecognition sites are often the target of such artificial manipulations,but other site specific targets, e.g., promoters, DNA replication sites,regulation sequences, control sequences, or other useful features may beincorporated by design. A similar concept is intended for a recombinant,e.g., fusion, polypeptide. Specifically included are synthetic nucleicacids which, by genetic code redundancy, encode polypeptides similar tofragments of these antigens, and fusions of sequences from variousdifferent species variants.

[0065] A significant “fragment” in a nucleic acid context is acontiguous segment of at least about 17 nucleotides, generally at least20 nucleotides, more generally at least 23 nucleotides, ordinarily atleast 26 nucleotides, more ordinarily at least 29 nucleotides, often atleast 32 nucleotides, more often at least 35 nucleotides, typically atleast 38 nucleotides more typically at least 41 nucleotides, usually atleast 44 nucleotides, more usually at least 47 nucleotides, preferablyat least 50 nucleotides, more preferably at least 53 nucleotides, and inparticularly preferred embodiments will be at least 56 or morenucleotides. Said fragments may have termini at any location, butespecially at boundaries between structural domains.

[0066] A DNA which codes for a CTLA-8 protein will be particularlyuseful to identify genes, mRNA, and cDNA species which code for relatedor homologous proteins, as well as DNAs which code for homologousproteins from different species. There are likely homologues in otherspecies, including primates. Various CTLA-8 proteins should behomologous and ate encompassed herein. However, even proteins that havea more distant evolutionary relationship to the antigen can readily beisolated under appropriate conditions using these sequences if they aresufficiently homologous. Primate CTLA-8 protein proteins are ofparticular interest.

[0067] This invention further covers recombinant DNA molecules andfragments having a DNA sequence identical to or highly homologous to theisolated DNAs set forth herein. In particular, the sequences will oftenbe operably linked to DNA segments which control transcription,translation, and DNA replication. Alternatively, recombinant clonesderived from the genomic sequences, e.g., containing introns, will beuseful for transgenic studies, including, e.g., transgenic cells andorganisms, and for gene therapy. See, e.g., Goodnow (1992) “TransgenicAnimals” in Roitt (ed.) Encyclopedia of Immunology Academic Press, SanDiego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al.(1991) Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson(1987)(ed.) Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach IRL Press, Oxford; Rosenberg (1992) J. Clinical Oncology10:180-199; and Cournoyer and Caskey (1993) Ann. Rev. Immunol.11:297-329.

[0068] Homologous nucleic acid sequences, when compared, exhibitsignificant similarity. The standards for homology in nucleic acids areeither measures for homology generally used in the art by sequencecomparison or based upon hybridization conditions. The hybridizationconditions are described in greater detail below.

[0069] Substantial homology in the nucleic acid sequence comparisoncontext means either that the segments, or their complementary strands,when compared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least 56%, more generally at least 59%,ordinarily at least 62%, more ordinarily at least 65%, often at least68%, more often at least 71%, typically at least 74%, more typically atleast 77%, usually at least 80%, more usually at least about 85%,preferably at least about 90%, more preferably at least about 95 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to astrand, or its complement, typically using a sequence derived from Table1, 2, or 3. Typically, selective hybridization will occur when there isat least about 55% homology over a stretch of at least about 14nucleotides, preferably at least about 65%, more preferably at leastabout 75%, and most preferably at least about 90%. See, Kanehisa (1984)Nuc. Acids Res. 12:203-213. The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willbe over a stretch of at least about 17 nucleotides, usually at leastabout 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about40 nucleotides, preferably at least about 50 nucleotides, and morepreferably at least about 75 to 100 or more nucleotides.

[0070] Stringent conditions, in referring to homology in thehybridization context, will be stringent combined conditions of salt,temperature, organic solvents, and other parameters, typically thosecontrolled in hybridization reactions. Stringent temperature conditionswill usually include temperatures in excess of about 30° C., moreusually in excess of about 37° C., typically in excess of about 45° C.,more typically in excess of about 55° C., preferably in excess of about65° C., and more preferably in excess of about 70° C. Stringent saltconditions will ordinarily be less than about 1000 mM, usually less thanabout 500 mM, more usually less than about 400 mM, typically less thanabout 300 mM, preferably less than about 200 mM, and more preferablyless than about 150 mM. However, the combination of parameters is muchmore important than the measure of any single parameter. See, e.g.,Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.

[0071] CTLA-8 protein from other mammalian species can be cloned andisolated by cross-species hybridization of closely related species,e.g., human, as disclosed in Table 3. Homology may be relatively lowbetween distantly related species, and thus hybridization of relativelyclosely related species is advisable. Alternatively, preparation of anantibody preparation which exhibits less species specificity may beuseful in expression cloning approaches.

[0072] III. Purified CTLA-8 Protein

[0073] The predicted sequence of murine CTLA-8 protein amino acidsequence is shown in Table 1. The homologous herpesvirus predicted ORF13protein sequence is shown in Table 2, and is assigned SEQ ID NO: 4. Ahuman counterpart is described in Table 3. The peptide sequences allowpreparation of peptides to generate antibodies to recognize suchsegments.

[0074] As used herein, the terms “murine CTLA-8 protein” and “humanCTLA-8 protein shall encompass, when used in a protein context, aprotein having amino acid sequences shown in Table 1 or Table 3, or asignificant fragment of such a protein. It also refers to a mousederived polypeptide which exhibits similar biological function orinteracts with CTLA-8 protein specific binding components. These bindingcomponents, e.g., antibodies, typically bind to a CTLA-8 protein withhigh affinity, e.g., at least about 100 nM, usually better than about 30nM, preferably better than about 10 nM, and more preferably at betterthan about 3 nM. Homologous proteins would be found in mammalian speciesother than rat or humans, e.g., mouse, primates, and in the herpesvirusgenome, e.g., ORF13. Non-mammalian species should also possessstructurally or functionally related genes and proteins.

[0075] The term “polypeptide” as used herein includes a significantfragment or segment, and encompasses a stretch of amino acid residues ofat least about 8 amino acids, generally at least 10 amino acids, moregenerally at least 12 amino acids, often at least 14 amino acids, moreoften at least 16 amino acids, typically at least 18 amino acids, moretypically at least 20 amino acids, usually at least 22 amino acids, moreusually at least 24 amino acids, preferably at least 26 amino acids,more preferably at least 28 amino acids, and, in particularly preferredembodiments, at least about 30 or more amino acids. The specific ends ofsuch a segment will be at any combinations within the protein,preferably encompassing structural domains.

[0076] The term “binding composition” refers to molecules that bind withspecificity to CTLA-8 protein, e.g., in a ligand-receptor type fashion,an antibody-antigen interaction, or compounds, e.g., proteins whichspecifically associate with CTLA-8 protein, e.g., in a naturalphysiologically relevant protein-protein interaction, either covalent ornon-covalent. The molecule may be a polymer, or chemical reagent. Noimplication as to whether CTLA-8 protein is either the ligand or thereceptor of a ligand-receptor interaction is represented, other than theinteraction exhibit similar specificity, e.g., specific affinity. Afunctional analog may be a protein with structural modifications, or maybe a wholly unrelated molecule, e.g., which has a molecular shape whichinteracts with the appropriate binding determinants. The proteins mayserve as agonists or antagonists of a receptor, see, e.g., Goodman, etal. (eds.) (1990) Goodman & Gilman's: The Pharmacological Bases ofTherapeutics (8th ed.), Pergamon Press.

[0077] Solubility of a polypeptide or fragment depends upon theenvironment and the polypeptide. Many parameters affect polypeptidesolubility, including temperature, electrolyte environment, size andmolecular characteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. and more usually greater than about 22° C. Fordiagnostic purposes, the temperature will usually be about roomtemperature or warmer, but less than the denaturation temperature ofcomponents in the assay. For therapeutic purposes, the temperature willusually be body temperature, typically about 37° C. for humans, thoughunder certain situations the temperature may be raised or lowered insitu or in vitro.

[0078] The electrolytes will usually approximate in situ physiologicalconditions, but may be modified to higher or lower ionic strength whereadvantageous. The actual ions may be modified, e.g., to conform tostandard buffers used in physiological or analytical contexts.

[0079] The size and structure of the polypeptide should generally be ina substantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents in a manner which approximates natural lipid bilayerinteractions.

[0080] The solvent will usually be a biologically compatible buffer, ofa type used for preservation of biological activities, and will usuallyapproximate a physiological solvent. Usually the solvent will have aneutral pH, typically between about 5 and 10, and preferably about 7.5.On some occasions, a detergent will be added, typically a mildnon-denaturing one, e.g., CHS or CHAPS, or a low enough concentration asto avoid significant disruption of structural or physiologicalproperties of the antigen.

[0081] Solubility is reflected by sedimentation measured in Svedbergunits, which are a measure of the sedimentation velocity of a moleculeunder particular conditions. The determination of the sedimentationvelocity was classically performed in an analytical ultracentrifuge, butis typically now performed in a standard ultracentrifuge. See,Freifelder (1982) Physical Biochemistry (2d ed.), W. H. Freeman; andCantor and Schimmel (1980) Biophysical Chemistry, parts 1-3, W. H.Freeman & Co., San Francisco. As a crude determination, a samplecontaining a putatively soluble polypeptide is spun in a standard fullsized ultracentrifuge at about 50K rpm for about 10 minutes, and solublemolecules will remain in the supernatant. A soluble particle orpolypeptide will typically be less than about 30 S, more typically lessthan about 15 S, usually less than about 10 S, more usually less thanabout 6 S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3 S.

[0082] IV. Making CTLA-8 Protein; Mimetics

[0083] DNA which encodes the CTLA-8 protein or fragments thereof can beobtained by chemical synthesis, screening cDNA libraries, or byscreening genomic libraries prepared from a wide variety of cell linesor tissue samples.

[0084] This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length protein or fragments which can in turn, forexample, be used to generate polyclonal or monoclonal antibodies; forbinding studies; for construction and expression of modified molecules;and for structure/function studies. Each antigen or its fragments can beexpressed in host cells that are transformed or transfected withappropriate expression vectors. These molecules can be substantiallypurified to be free of protein or cellular contaminants, other thanthose derived from the recombinant host, and therefore are particularlyuseful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The antigen, orportions thereof, may be expressed as fusions with other proteins.

[0085] Expression vectors are typically self-replicating DNA or RNAconstructs containing the desired antigen gene or its fragments, usuallyoperably linked to suitable genetic control elements that are recognizedin a suitable host cell. These control elements are capable of effectingexpression within a suitable host. The specific type of control elementsnecessary to effect expression will depend upon the eventual host cellused. Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently of thehost cell. Methods for amplifying vector copy number are also known,see, e.g., Kaufman, et al. (1985) Molec. and Cell. Biol. 5:1750-1759.

[0086] The vectors of this invention contain DNA which encodes a CTLA-8protein, or a fragment thereof, typically encoding a biologically activepolypeptide. The DNA can be under the control of a viral promoter andcan encode a selection marker. This invention further contemplates useof such expression vectors which are capable of expressing eukaryoticcDNA coding for a CTLA-8 protein in a prokaryotic or eukaryotic host,where the vector is compatible with the host and where the eukaryoticcDNA coding for the antigen is inserted into the vector such that growthof the host containing the vector expresses the cDNA in question.Usually, expression vectors are designed for stable replication in theirhost cells or for amplification to greatly increase the total number ofcopies of the desirable gene per cell. It is not always necessary torequire that an expression vector replicate in a host cell, e.g., it ispossible to effect transient expression of the antigen or its fragmentsin various hosts using vectors that do not contain a replication originthat is recognized by the host cell. It is also possible to use vectorsthat cause integration of a CTLA-8 protein gene or its fragments intothe host DNA by recombination, or to integrate a promoter which controlsexpression of an endogenous gene.

[0087] Vectors, as used herein, comprise plasmids, viruses,bacteriophage, integratable DNA fragments, and other vehicles whichenable the integration of DNA fragments into the genome of the host.Expression vectors are specialized vectors which contain genetic controlelements that effect expression of operably linked genes. Plasmids arethe most commonly used form of vector but all other forms of vectorswhich serve an equivalent function and which are, or become, known inthe art are suitable for use herein. See, e.g., Pouwels, et al.. (1985and Supplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,and Rodriquez, et al. (1988)(eds.) Vectors: A Survey of MolecularCloning Vectors and Their Uses, Buttersworth, Boston, Mass.

[0088] Transformed cells include cells, preferably mammalian, that havebeen transformed or transfected with vectors containing a CTLA-8 gene,typically constructed using recombinant DNA techniques. Transformed hostcells usually express the antigen or its fragments, but for purposes ofcloning, amplifying, and manipulating its DNA, do not need to expressthe protein. This invention further contemplates culturing transformedcells in a nutrient medium, thus permitting the protein to accumulate inthe culture. The protein can be recovered, either from the culture orfrom the culture medium.

[0089] For purposes of this invention, DNA sequences are operably linkedwhen they are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression.

[0090] Suitable host cells include prokaryotes, lower eukaryotes, andhigher eukaryotes. Prokaryotes include both gram negative and grampositive organisms, e.g., E. coli and B. subtilis. Lower eukaryotesinclude yeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

[0091] Prokaryotic host-vector systems include a wide variety of vectorsfor many different species. As used herein, E. coli and its vectors willbe used generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 ormany of its derivatives. Vectors that can be used to express the CTLA-8proteins or its fragments include, but are not limited to, such vectorsas those containing the lac promoter (pUC-series); trp promoter(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters(pOTS); or hybrid promoters such as ptac (pDR540). See Brosius, et al.(1988) “Expression Vectors Employing Lambda-, trp-, lac-, andIpp-derived Promoters”, in Rodriguez and Denhardt (eds.) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Chapter 10, pp. 205-236.

[0092] Lower eukaryotes, e.g., yeasts and Dictyostelium, may betransformed with vectors encoding CTLA-8 proteins. For purposes of thisinvention, the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used to generically represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the desired protein or its fragments, and sequences fortranslation termination, polyadenylation, and transcription termination.Suitable expression vectors for yeast include such constitutivepromoters as 3-phosphoglycerate kinase and various other glycolyticenzyme gene promoters or such inducible promoters as the alcoholdehydrogenase 2 promoter or metallothionine promoter. Suitable vectorsinclude derivatives of the following types: self-replicating low copynumber (such as the YRp-series), self-replicating high copy number (suchas the YEp-series); integrating types (such as the YIp-series), ormini-chromosomes (such as the YCp-series).

[0093] Higher eukaryotic tissue culture cells are the preferred hostcells for expression of the functionally active CTLA-8 protein. Inprinciple, many higher eukaryotic tissue culture cell lines areworkable, e.g., insect baculovirus expression systems, whether from aninvertebrate or vertebrate source. However, mammalian cells arepreferred, in that the processing, both cotranslationally andposttranslationally. Transformation or transfection and propagation ofsuch cells has become a routine procedure. Examples of useful cell linesinclude HeLa cells, Chinese hamster ovary (CHO) cell lines, baby ratkidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey(COS) cell lines. Expression vectors for such cell lines usually includean origin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pCDNA1; pCD, see Okayama, et al. (1985) Mol.Cell Biol. 5:1136-1142; pMC1neo Poly-A, see Thomas, et al. (1987) Cell51:503-512; and a baculovirus vector such as pAC 373 or pAC 610, seeO'Reilly, et al. (1992) Baculovirus Expression Vectors: A LaboratoryManual Freeman and Co., CRC Press, Boca Raton, Fla.

[0094] It will often be desired to express a CTLA-8 protein polypeptidein a system which provides a specific or defined glycosylation pattern.In this case, the usual pattern will be that provided naturally by theexpression system. However, the pattern will be modifiable by exposingthe polypeptide, e.g., an unglycosylated form, to appropriateglycosylating proteins introduced into a heterologous expression system.For example, the CTLA-8 protein gene may be co-transformed with one ormore genes encoding mammalian or other glycosylating enzymes. Using thisapproach, certain mammalian glycosylation patterns will be achievable orapproximated in prokaryote or other cells.

[0095] The CTLA-8 protein, or a fragment thereof, may be engineered tobe phosphatidyl inositol (PI) linked to a cell membrane, but can beremoved from membranes by treatment with a phosphatidyl inositolcleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. Thisreleases the antigen in a biologically active form, and allowspurification by standard procedures of protein chemistry. See, e.g., Low(1989) Biochim. Biophys. Acta 988:427-454; Tse, et al. (1985) Science230:1003-1008; and Brunner, et al. (1991) J. Cell Biol. 114:1275-1283.

[0096] Now that the CTLA-8 protein has been characterized, fragments orderivatives thereof can be prepared by conventional processes forsynthesizing peptides. These include processes such as are described inStewart and Young (1984) Solid Phase Peptide Synthesis, Pierce ChemicalCo., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice ofPeptide Synthesis, Springer-Verlag, New York; and Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer-Verlag, New York. For example,an azide process, an acid chloride process, an acid anhydride process, amixed anhydride process, an active ester process (for example,p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester),a carbodiimidazole process, an oxidative-reductive process, or adicyclohexylcarbodiimide (DCCD)/additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses.

[0097] The CTLA-8 protein, fragments, or derivatives are suitablyprepared in accordance with the above processes as typically employed inpeptide synthesis, generally either by a so-called stepwise processwhich comprises condensing an amino acid to the terminal amino acid, oneby one in sequence, or by coupling peptide fragments to the terminalamino acid. Amino groups that are not being used in the couplingreaction are typically protected to prevent coupling at an incorrectlocation.

[0098] If a solid phase synthesis is adopted, the C-terminal amino acidis bound to an insoluble carrier or support through its carboxyl group.The insoluble carrier is not particularly limited as long as it has abinding capability to a reactive carboxyl group. Examples of suchinsoluble carriers include halomethyl resins, such as chloromethyl resinor bromomethyl resin, hydroxymethyl resins, phenol resins,tert-alkyloxycarbonyl-hydrazidated resins, and the like.

[0099] An amino group-protected amino acid is bound in sequence throughcondensation of its activated carboxyl group and the reactive aminogroup of the previously formed peptide or chain, to synthesize thepeptide step by step. After synthesizing the complete sequence, thepeptide is split off from the insoluble carrier to produce the peptide.This solid-phase approach is generally described by Merrifield, et al.(1963) in J. Am. Chem. Soc. 85:2149-2156.

[0100] The prepared protein and fragments thereof can be isolated andpurified from the reaction mixture by means of peptide separation, forexample, by extraction, precipitation, electrophoresis and various formsof chromatography, and the like. The CTLA-8 proteins of this inventioncan be obtained in varying degrees of purity depending upon its desireduse. Purification can be accomplished by use of the protein purificationtechniques disclosed herein or by the use of the antibodies hereindescribed in immunoabsorbant affinity chromatography. Thisimmunoabsorbant affinity chromatography is carried out by first linkingthe antibodies to a solid support and then contacting the linkedantibodies with solubilized lysates of appropriate source cells, lysatesof other cells expressing the protein, or lysates or supernatants ofcells producing the CTLA-8 protein as a result of DNA techniques, seebelow.

[0101] V. Physical Variants

[0102] This invention also encompasses proteins or peptides havingsubstantial amino acid sequence homology with the amino acid sequence ofthe CTLA-8 protein. The variants include species or allelic variants.

[0103] Amino acid sequence homology, or sequence identity, is determinedby optimizing residue matches, if necessary, by introducing gaps asrequired. This changes when considering conservative substitutions asmatches. Conservative substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Homologousamino acid sequences are typically intended to include natural allelicand interspecies variations in each respective protein sequence. Typicalhomologous proteins or peptides will have from 25-100% homology (if gapscan be introduced), to 50-100% homology (if conservative substitutionsare included) with the amino acid sequence of the CTLA-8 protein.Homology measures will be at least about 35%, generally at least 40%,more generally at least 45%, often at least 50%, more often at least55%, typically at least 60%, more typically at least 65%, usually atleast 70%, more usually at least 75%, preferably at least 80%, and morepreferably at least 80%, and in particularly preferred embodiments, atleast 85% or more. See also Needleham, et al. (1970) J. Mol. Biol.48:443-453; Sankoff, et al. (1983) Chapter One in Time Warps, StringEdits, and Macromolecules: The Theory and Practice of SequenceComparison Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis.

[0104] The isolated DNA encoding a CTLA-8 protein can be readilymodified by nucleotide substitutions, nucleotide deletions, nucleotideinsertions, and inversions of nucleotide stretches. These modificationsresult in novel DNA sequences which encode these antigens, theirderivatives, or proteins having similar physiological, immunogenic, orantigenic activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. Such mutant CTLA-8 proteinderivatives include predetermined or site-specific mutations of therespective protein or its fragments. “Mutant CTLA-8 protein” encompassesa polypeptide otherwise falling within the homology definition of themurine CTLA-8 or human CTLA-8 protein as set forth above, but having anamino acid sequence which differs from that of CTLA-8 protein as foundin nature, whether by way of deletion, substitution, or insertion. Inparticular, “site specific mutant CTLA-8 protein” generally includesproteins having significant homology with a protein having sequences ofTable 1, 2, or 3, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the disclosed sequences. Similar conceptsapply to different CTLA-8 proteins, particularly those found in variouswarm blooded animals, e.g., mammals and birds. As stated before, it isemphasized that descriptions are generally meant to encompass all CTLA-8proteins, not limited to the mouse embodiment specifically discussed.

[0105] Although site specific mutation sites are predetermined, mutantsneed not be site specific. CTLA-8 protein mutagenesis can be conductedby making amino acid insertions or deletions. Substitutions, deletions,insertions, or any combinations may be generated to arrive at a finalconstruct. Insertions include amino- or carboxy-terminal fusions. Randommutagenesis can be conducted at a target codon and the expressed mutantscan then be screened for the desired activity. Methods for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known in the art, e.g., by M13 primer mutagenesis orpolymerase chain reaction (PCR) techniques. See also Sambrook, et al.(1989) and Ausubel, et al. (1987 and Supplements).

[0106] The mutations in the DNA normally should not place codingsequences out of reading frames and preferably will not createcomplementary regions that could hybridize to produce secondary mRNAstructure such as loops or hairpins.

[0107] The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of an immunoglobulin with a CTLA-8 polypeptide is a continuousprotein molecule having sequences fused in a typical peptide linkage,typically made as a single translation product and exhibiting propertiesderived from each source peptide. A similar concept applies toheterologous nucleic acid sequences.

[0108] In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, antigen-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science,243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.Thus, new chimeric polypeptides exhibiting new combinations ofspecificities will result from the functional linkage of biologicallyrelevant domains and other functional domains.

[0109] The phosphoramidite method described by Beaucage and Carruthers(1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

[0110] VI. Functional Variants

[0111] The blocking of physiological response to CTLA-8 proteins mayresult from the inhibition of binding of the antigen to its naturalbinding partner, e.g., through competitive inhibition. Thus, in vitroassays of the present invention will often use isolated protein,membranes from cells expressing a recombinant membrane associated CTLA-8protein, soluble fragments comprising binding segments, or fragmentsattached to solid phase substrates. These assays will also allow for thediagnostic determination of the effects of either binding segmentmutations and modifications, or protein mutations and modifications,e.g., analogs.

[0112] This invention also contemplates the use of competitive drugscreening assays, e.g., where neutralizing antibodies to antigen orbinding partner fragments compete with a test compound for binding tothe protein. In this manner, the antibodies can be used to detect thepresence of any polypeptide which shares one or more antigenic bindingsites of the protein and can also be used to occupy binding sites on theprotein that might otherwise interact with a binding partner.

[0113] Additionally, neutralizing antibodies against the CTLA-8 proteinand soluble fragments of the antigen which contain a high affinityreceptor binding site, can be used to inhibit antigen function intissues, e.g., tissues experiencing abnormal physiology.

[0114] “Derivatives” of the CTLA-8 antigens include amino acid sequencemutants, glycosylation variants, and covalent or aggregate conjugateswith other chemical moieties. Covalent derivatives can be prepared bylinkage of functionalities to groups which are found in the CTLA-8 aminoacid side chains or at the N- or C-termini, by means which are wellknown in the art. These derivatives can include, without limitation,aliphatic esters or amides of the carboxyl terminus, or of residuescontaining carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino terminalamino acid or amino-group containing residues, e.g., lysine or arginine.Acyl groups are selected from the group of alkyl-moieties including C3to C18 normal alkyl, thereby forming alkanoyl aroyl species. Covalentattachment to carrier proteins may be important when immunogenicmoieties are haptens.

[0115] In particular, glycosylation alterations are included, e.g., madeby modifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

[0116] A major group of derivatives are covalent conjugates of theCTLA-8 protein or fragments thereof with other proteins or polypeptides.These derivatives can be synthesized in recombinant culture such as N-or C-terminal fusions or by the use of agents known in the art for theirusefulness in cross-linking proteins through reactive side groups.Preferred antigen derivatization sites with cross-linking agents are atfree amino groups, carbohydrate moieties, and cysteine residues.

[0117] Fusion polypeptides between the CTLA-8 proteins and otherhomologous or heterologous proteins are also provided. Homologouspolypeptides may be fusions between different surface markers, resultingin, e.g., a hybrid protein exhibiting receptor binding specificity.Likewise, heterologous fusions may be constructed which would exhibit acombination of properties or activities of the derivative proteins.Typical examples are fusions of a reporter polypeptide, e.g.,luciferase, with a segment or domain of an antigen, e.g., areceptor-binding segment, so that the presence or location of the fusedantigen may be easily determined. See, e.g., Dull, et al., U.S. Pat. No.4,859,609. Other gene fusion partners include bacterial β-galactosidase,trpE, Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase, andyeast alpha mating factor. See, e.g., Godowski, et al. (1988) Science241:812-816.

[0118] The phosphoramidite method described by Beaucage and Carruthers(1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

[0119] Such polypeptides may also have amino acid residues which havebeen chemically modified by phosphorylation, sulfonation, biotinylation,or the addition or removal of other moieties, particularly those whichhave molecular shapes similar to phosphate groups. In some embodiments,the modifications will be useful labeling reagents, or serve aspurification targets, e.g., affinity ligands.

[0120] Fusion proteins will typically be made by either recombinantnucleic acid methods or by synthetic polypeptide methods. Techniques fornucleic acid manipulation and expression are described generally, forexample, in Sambrook, et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed.), Vols. 1-3, Cold Spring Harbor Laboratory. Techniquesfor synthesis of polypeptides are described, for example, in Merrifield(1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232:341-347; and Atherton, et al. (1989) Solid Phase Peptide Synthesis: APractical Approach, IRL Press, Oxford.

[0121] This invention also contemplates the use of derivatives of theCTLA-8 proteins other than variations in amino acid sequence orglycosylation. Such derivatives may involve covalent or aggregativeassociation with chemical moieties. These derivatives generally fallinto the three classes: (1) salts, (2) side chain and terminal residuecovalent modifications, and (3) adsorption complexes, for example withcell membranes. Such covalent or aggregative derivatives are useful asimmunogens, as reagents in immunoassays, or in purification methods suchas for affinity purification of antigens or other binding proteins. Forexample, a CTLA-8 antigen can be immobilized by covalent bonding to asolid support such as cyanogen bromide-activated Sepharose, by methodswhich are well known in the art, or adsorbed onto polyolefin surfaces,with or without glutaraldehyde cross-linking, for use in the assay orpurification of anti-CTLA-8 protein antibodies or its receptor or otherbinding partner. The CTLA-8 antigens can also be labeled with adetectable group, for example radioiodinated by the chloramine Tprocedure, covalently bound to rare earth chelates, or conjugated toanother fluorescent moiety for use in diagnostic assays. Purification ofCTLA-8 protein may be effected by immobilized antibodies or bindingpartners.

[0122] A solubilized CTLA-8 antigen or fragment of this invention can beused as an immunogen for the production of antisera or antibodiesspecific for the protein or fragments thereof. The purified antigen canbe used to screen monoclonal antibodies or binding fragments prepared byimmunization with various forms of impure preparations containing theprotein. In particular, the term “antibodies” also encompasses antigenbinding fragments of natural antibodies. The purified CTLA-8 proteinscan also be used as a reagent to detect any antibodies generated inresponse to the presence of elevated levels of the protein or cellfragments containing the antigen, both of which may be diagnostic of anabnormal or specific physiological or disease condition. Additionally,antigen fragments may also serve as immunogens to produce the antibodiesof the present invention, as described immediately below. For example,this invention contemplates antibodies raised against amino acidsequences encoded by nucleotide sequences shown in Table 1, 2, or 3, orfragments of proteins containing them. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific fragments which are predicted to lie outside of thelipid bilayer.

[0123] The present invention contemplates the isolation of additionalclosely related species variants. Southern blot analysis establishedthat similar genetic entities exist in other mammals, e.g., rat andhuman. It is likely that the CTLA-8 proteins are widespread in speciesvariants, e.g., rodents, lagomorphs, carnivores, artiodactyla,perissodactyla, and primates.

[0124] The invention also provides means to isolate a group of relatedantigens displaying both distinctness and similarities in structure,expression, and function. Elucidation of many of the physiologicaleffects of the antigens will be greatly accelerated by the isolation andcharacterization of distinct species variants. In particular, thepresent invention provides useful probes for identifying additionalhomologous genetic entities in different species.

[0125] The isolated genes will allow transformation of cells lackingexpression of a corresponding CTLA-8 protein, e.g., either species typesor cells which lack corresponding antigens and should exhibit negativebackground activity. Expression of transformed genes will allowisolation of antigenically pure cell lines, with defined or singlespecie variants. This approach will allow for more sensitive detectionand discrimination of the physiological effects of CTLA-8 proteins.Subcellular fragments, e.g., cytoplasts or membrane fragments, can beisolated and used.

[0126] Dissection of the critical structural elements which effect thevarious physiological or differentiation functions provided by theproteins is possible using standard techniques of modern molecularbiology, particularly in comparing members of the related class. See,e.g., the homolog-scanning mutagenesis technique described inCunningham, et al. (1989) Science 243:1339-1336; and approaches used inO'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, etal. (1990) EMBO J. 9:4381-4390.

[0127] In particular, functional domains or segments can be substitutedbetween species variants to determine what structural features areimportant in both binding partner affinity and specificity, as well assignal transduction. An array of different variants will be used toscreen for molecules exhibiting combined properties of interaction withdifferent species variants of binding partners.

[0128] Antigen internalization may occur under certain circumstances,and interaction between intracellular components and “extracellular”segments of proteins involved in interactions may occur. The specificsegments of interaction of CTLA-8 protein with other intracellularcomponents may be identified by mutagenesis or direct biochemical means,e.g., cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of biological function willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

[0129] Further study of the expression and control of CTLA-8 proteinwill be pursued. The controlling elements associated with the antigensmay exhibit differential developmental, tissue specific, or otherexpression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest.

[0130] Structural studies of the antigen will lead to design of newvariants, particularly analogs exhibiting agonist or antagonistproperties on binding partners. This can be combined with previouslydescribed screening methods to isolate variants exhibiting desiredspectra of activities.

[0131] Expression in other cell types will often result in glycosylationdifferences in a particular antigen. Various species variants mayexhibit distinct functions based upon structural differences other thanamino acid sequence. Differential modifications may be responsible fordifferential function, and elucidation of the effects are now madepossible.

[0132] Thus, the present invention provides important reagents relatedto antigen-binding partner interaction. Although the foregoingdescription has focused primarily upon the murine CTLA-8 and humanCTLA-8 protein, those of skill in the art will immediately recognizethat the invention encompasses other antigens, e.g., mouse and othermammalian species or allelic variants, as well as variants thereof.

[0133] VII. Antibodies

[0134] Antibodies can be raised to the various CTLA-8 proteins,including species or allelic variants, and fragments thereof, both intheir naturally occurring forms and in their recombinant forms.Additionally, antibodies can be raised to CTLA-8 proteins in eithertheir active forms or in their inactive forms. Anti-idiotypic antibodiesare also contemplated.

[0135] Antibodies, including binding fragments and single chainversions, against predetermined fragments of the antigens can be raisedby immunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective CTLA-8 proteins, or screened foragonistic or antagonistic activity, e.g., mediated through a bindingpartner. These monoclonal antibodies will usually bind with at least aK_(D) of about 1 mM, more usually at least about 300 μM, typically atleast about 10 μm, more typically at least about 30 μM, preferably atleast about 10 μm, and more preferably at least about 3 μm or better.

[0136] The antibodies, including antigen binding fragments, of thisinvention can have significant diagnostic or therapeutic value. They canbe potent antagonists that bind to a binding partner and inhibit antigenbinding or inhibit the ability of an antigen to elicit a biologicalresponse. They also can be useful as non-neutralizing antibodies and canbe coupled to toxins or radionuclides so that when the antibody binds tothe antigen, a cell expressing it, e.g., on its surface, is killed.Further, these antibodies can be conjugated to drugs or othertherapeutic agents, either directly or indirectly by means of a linker,and may effect drug targeting.

[0137] The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingby a partner. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying CTLA-8 protein or its binding partners. See, e.g., Chan(ed.)(1987) Immunoassay: A Practical Guide Academic Press, Orlando,Fla.; Ngo (ed.)(1988) Nonisotopic Immunoassay Plenum Press, NY; andPrice and Newman (eds.)(1991) Principles and Practice of ImmunoassayStockton Press, NY.

[0138] Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York, and Williams,et. al. (1967) Methods in Immunology and Immunochemistry, Vol. 1,Academic Press, New York, for descriptions of methods of preparingpolyclonal antisera. A typical method involves hyperimmunization of ananimal with an antigen. The blood of the animal is then collectedshortly after the repeated immunizations and the gamma globulin isisolated.

[0139] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basicand Clinical Immunology (4th ed.), Lange Medical Publications, LosAltos, Calif., and references cited therein; Harlow and Lane (1988)Antibodies: A Laboratory Manual, CSH Press; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.) Academic Press, New York;and particularly in Kohler and Milstein (1975) in Nature 256: 495-497,which discusses one method of generating monoclonal antibodies.Summarized briefly, this method involves injecting an animal with animmunogen. The animal is then sacrificed and cells taken from itsspleen, which are then fused with myeloma cells. The result is a hybridcell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secrete a single antibody species to the immunogen. Inthis manner, the individual antibody species obtained are the productsof immortalized and cloned single B cells from the immune animalgenerated in response to a specific site recognized on the immunogenicsubstance.

[0140] Other suitable techniques involve in vitro exposure oflymphocytes to the antigenic polypeptides or alternatively to selectionof libraries of antibodies in phage or similar vectors. See, Huse, etal. (1989) “Generation of a Large Combinatorial Library of theImmunoglobulin Repertoire in Phage Lambda,” Science 246:1275-1281; andWard, et al. (1989) Nature 341:544-546. The polypeptides and antibodiesof the present invention may be used with or without modification,including chimeric or humanized antibodies. Frequently, the polypeptidesand antibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567.

[0141] The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support, e.g., particles, such asagarose, Sephadex, or the like, where a cell lysate may be passedthrough the column, the column washed, followed by increasingconcentrations of a mild denaturant, whereby the purified CTLA-8 proteinwill be released.

[0142] The antibodies-may also be used to screen expression librariesfor particular expression products. Usually the antibodies used in sucha procedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

[0143] Antibodies raised against each CTLA-8 protein will also be usefulto raise anti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

[0144] VIII. Uses

[0145] The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for physiological or developmental abnormalities, orbelow in the description of kits for diagnosis.

[0146] This invention also provides reagents with significanttherapeutic value. The CTLA-8 protein (naturally occurring orrecombinant), fragments thereof, and antibodies thereto, along withcompounds identified as having binding affinity to CTLA-8 protein,should be useful in the treatment of conditions associated with abnormalphysiology or development, including abnormal proliferation, e.g.,cancerous conditions, or degenerative conditions. Abnormalproliferation, regeneration, degeneration, and atrophy may be modulatedby appropriate therapeutic treatment using the compositions providedherein. For example, a disease or disorder associated with abnormalexpression or abnormal signaling by a CTLA-8 antigen should be a likelytarget for an agonist or antagonist of the protein.

[0147] Other abnormal developmental conditions are known in the celltypes shown to possess CTLA-8 antigen mRNA by Northern blot analysis.See Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co.,Rahway, N.J.; and Thorn, et al. Harrison's Principles of InternalMedicine, McGraw-Hill, N.Y. These. problems may be susceptible toprevention or treatment using compositions provided herein.

[0148] Recombinant antibodies which bind to CTLA-8 can be purified andthen administered to a patient. These reagents can be combined fortherapeutic use with additional active or inert ingredients, e.g., inconventional pharmaceutically acceptable carriers or diluents, e.g.,immunogenic adjuvants, along with physiologically innocuous stabilizersand excipients. These combinations can be sterile filtered and placedinto dosage forms as by lyophilization in dosage vials or storage instabilized aqueous preparations. This invention also contemplates use ofantibodies or binding fragments thereof, including forms which are notcomplement binding.

[0149] Screening using CTLA-8 for binding partners or compounds havingbinding affinity to CTLA-8 antigen can be performed, including isolationof associated components. Subsequent biological assays can then beutilized to determine if the compound has intrinsic biological activityand is therefore an agonist or antagonist in that it blocks an activityof the antigen. This invention further contemplates the therapeutic useof antibodies to CTLA-8 protein as antagonists. This approach should beparticularly useful with other CTLA-8 protein species variants.

[0150] The quantities of reagents necessary for effective therapy willdepend upon many different factors, including means of administration,target site, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. See also Langer (1990) Science 249:1527-1533.Pharmaceutically acceptable carriers will include water, saline,buffers, and other compounds described, e.g., in the Merck Index, Merck& Co., Rahway, N.J. Dosage ranges would ordinarily be expected to be inamounts lower than 1 mM concentrations, typically less than about 10 μmconcentrations, usually less than about 100 nM, preferably less thanabout 10 pM (picomolar), and most preferably less than about 1 fM(femtomolar), with an appropriate carrier. Slow release formulations, ora slow release apparatus will often be utilized for continuousadministration.

[0151] CTLA-8 protein, fragments thereof, and antibodies to it or itsfragments, antagonists, and agonists, may be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in any conventional dosage formulation. While it ispossible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press, Parrytown, N.Y.; Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, etal. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications 2ded., Dekker, N.Y.; Lieberman, et al. (eds.)(1990) Pharmaceutical DosageForms: Tablets 2d ed., Dekker, N.Y.; and Lieberman, et al. (eds.)(1990)Pharmaceutical Dosage Forms: Disperse Systems Dekker, N.Y. The therapyof this invention may be combined with or used in association with otherchemotherapeutic or chemopreventive agents.

[0152] Both the naturally occurring and the recombinant forms of theCTLA-8 proteins of this invention are particularly useful in kits andassay methods which are capable of screening compounds for bindingactivity to the proteins. Several methods of automating assays have beendeveloped in recent years so as to permit screening of tens of thousandsof compounds in a short period. See, e.g., Fodor, et al. (1991) Science251:767-773, which describes means for testing of binding affinity by aplurality of defined polymers synthesized on a solid substrate. Thedevelopment of suitable assays can be greatly facilitated by theavailability of large amounts of purified, soluble CTLA-8 protein asprovided by this invention.

[0153] This invention is particularly useful for screening compounds byusing recombinant antigen in any of a variety of drug screeningtechniques. The advantages of using a recombinant protein in screeningfor specific ligands include: (a) improved renewable source of theantigen from a specific source; (b) potentially greater number ofantigen molecules per cell giving better signal to noise ratio inassays; and (c) species variant specificity (theoretically givinggreater biological and disease specificity). The purified protein may betested in numerous assays, typically in vitro assays, which evaluatebiologically relevant responses. See, e.g., Coligan Current Protocols inImmunology; Hood, et al. Immunology Benjamin/Cummings; Paul (ed.)Fundamental Immunology; and Methods in Enzymology Academic Press.

[0154] One method of drug screening utilizes eukaryotic or prokaryotichost cells which are stably transformed with recombinant DNA moleculesexpressing the CTLA-8 antigens. Cells may be isolated which express anantigen in isolation from other functionally equivalent antigens. Suchcells, either in viable or fixed form, can be used for standardprotein-protein binding assays. See also, Parce, et al. (1989) Science246:243-247; and Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA87:4007-4011, which describe sensitive methods to detect cellularresponses. Competitive assays are particularly useful, where the cells(source of CTLA-8 protein) are contacted and incubated with a labeledbinding partner or antibody having known binding affinity to the ligand,such as ¹²⁵I-antibody, and a test sample whose binding affinity to thebinding composition is being measured. The bound and free labeledbinding compositions are then separated to assess the degree of antigenbinding. The amount of test compound bound is inversely proportional tothe amount of labeled receptor binding to the known source. Any one ofnumerous techniques can be used to separate bound from free antigen toassess the degree of binding. This separation step could typicallyinvolve a procedure such as adhesion to filters followed by washing,adhesion to plastic followed by washing, or centrifugation of the cellmembranes. Viable cells could also be used to screen for the effects ofdrugs on CTLA-8 protein mediated functions, e.g., second messengerlevels, i.e., Ca⁺⁺; cell proliferation; inositol phosphate pool changes;and others. Some detection methods allow for elimination of a separationstep, e.g., a proximity sensitive detection system. Calcium sensitivedyes will be useful for detecting Ca⁺⁺ levels, with a fluorimeter or afluorescence cell sorting apparatus.

[0155] Another method utilizes membranes from transformed eukaryotic orprokaryotic host cells as the source of the CTLA-8 protein. These cellsare stably transformed with DNA vectors directing the expression of amembrane associated CTLA-8 protein, e.g., an engineered membrane boundform. Essentially, the membranes would be prepared from the cells andused in any receptor/ligand type binding assay such as the competitiveassay set forth above.

[0156] Still another approach is to use solubilized, unpurified orsolubilized, purified CTLA-8 protein from transformed eukaryotic orprokaryotic host cells. This allows for a “molecular” binding assay withthe advantages of increased specificity, the ability to automate, andhigh drug test throughput.

[0157] Another technique for drug screening involves an approach whichprovides high throughput screening for compounds having suitable bindingaffinity to CTLA-8 and is described in detail in Geysen, European PatentApplication 84/03564, published on Sep. 13, 1984. First, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, e.g., plastic pins or some other appropriate surface, seeFodor, et al. (1991). Then all the pins are reacted with solubilized,unpurified or solubilized, purified CTLA-8 binding composition, andwashed. The next step involves detecting bound binding composition.

[0158] Rational drug design may also be based upon structural studies ofthe molecular shapes of the CTLA-8 protein and other effectors oranalogs. Effectors may be other proteins which mediate other functionsin response to antigen binding, or other proteins which normallyinteract with the antigen. One means for determining which sitesinteract with specific other proteins is a physical structuredetermination, e.g., x-ray crystallography or 2 dimensional NMRtechniques. These will provide guidance as to which amino acid residuesform molecular contact regions. For a detailed description of proteinstructural determination, see, e.g., Blundell and Johnson (1976) ProteinCrystallography, Academic Press, New York.

[0159] Purified CTLA-8 protein can be coated directly onto plates foruse in the aforementioned drug screening techniques. However,non-neutralizing antibodies to these ligands can be used as captureantibodies to immobilize the respective ligand on the solid phase.

[0160] IX. Kits

[0161] This invention also contemplates use of CTLA-8 proteins,fragments thereof, peptides, and their fusion products in a variety ofdiagnostic kits and methods for detecting the presence of a bindingcomposition. Typically the kit will have a compartment containing eithera defined CTLA-8 peptide or gene segment or a reagent which recognizesone or the other, e.g., antigen fragments or antibodies.

[0162] A kit for determining the binding affinity of a test compound toa CTLA-8 protein would typically comprise a test compound; a labeledcompound, for example an antibody having known binding affinity for theantigen; a source of CTLA-8 protein (naturally occurring orrecombinant); and a means for separating bound from free labeledcompound, such as a solid phase for immobilizing the antigen. Oncecompounds are screened, those having suitable binding affinity to theantigen can be evaluated in suitable biological assays, as are wellknown in the art, to determine whether they exhibit similar biologicalactivities to the natural antigen. The availability of recombinantCTLA-8 protein polypeptides also provide well defined standards forcalibrating such assays.

[0163] A preferred kit for determining the concentration of, forexample, a CTLA-8 protein in a sample would typically comprise a labeledcompound, e.g., antibody, having known binding affinity for the antigen,a source of antigen (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, for example, a solidphase for immobilizing the CTLA-8 protein. Compartments containingreagents, and instructions, will normally be provided.

[0164] One method for determining the concentration of CTLA-8 protein ina sample would typically comprise the steps of: (1) preparing membranesfrom a sample comprised of a membrane bound CTLA-8 protein source; (2)washing the membranes and suspending them in a buffer; (3) solubilizingthe antigen by incubating the membranes in a culture medium to which asuitable detergent has been added; (4) adjusting the detergentconcentration of the solubilized antigen; (5) contacting and incubatingsaid dilution with radiolabeled antibody to form complexes; (6)recovering the complexes such as by filtration through polyethyleneiminetreated filters; and (7) measuring the radioactivity of the recoveredcomplexes.

[0165] Antibodies, including antigen binding fragments, specific for theCTLA-8 protein or fragments are useful in diagnostic applications todetect the presence of elevated levels of CTLA-8 protein and/or itsfragments. Such diagnostic assays can employ lysates, live cells, fixedcells, immunofluorescence, cell cultures, body fluids, and further caninvolve the detection of antigens related to the protein in serum, orthe like. Diagnostic assays may be homogeneous (without a separationstep between free reagent and protein-protein complex) or heterogeneous(with a separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.For example, unlabeled antibodies can be employed by using a secondantibody which is labeled and which recognizes the antibody to a CTLA-8protein or to a particular fragment thereof. Similar assays have alsobeen extensively discussed in the literature. See, e.g., Harlow and Lane(1988) Antibodies: A Laboratory Manual, CSH.

[0166] Anti-idiotypic antibodies may have similar use to diagnosepresence of antibodies against a CTLA-8 protein, as such may bediagnostic of various abnormal states. For example, overproduction ofCTLA-8 protein may result in production of various immunologicalreactions which may be diagnostic of abnormal physiological states,particularly in proliferative cell conditions such as cancer or abnormaldifferentiation.

[0167] Frequently, the reagents for diagnostic assays are supplied inkits, so as to optimize the sensitivity of the assay. For the subjectinvention, depending upon the nature of the assay, the protocol, and thelabel, either labeled or unlabeled antibody, or labeled CTLA-8 proteinis provided. This is usually in conjunction with other additives, suchas buffers, stabilizers, materials necessary for signal production suchas substrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

[0168] Any of the aforementioned constituents of the drug screening andthe diagnostic assays may be used without modification or may bemodified in a variety of ways. For example, labeling may be achieved bycovalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In any of these assays, theantigen, test compound, CTLA-8 protein, or antibodies thereto can belabeled either directly or indirectly. Possibilities for direct labelinginclude label groups: radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No.3,645,090) such as peroxidase and alkaline phosphatase, and fluorescentlabels (U.S. Pat. No. 3,940,475) capable of monitoring the change influorescence intensity, wavelength shift, or fluorescence polarization.Possibilities for indirect labeling include biotinylation of oneconstituent followed by binding to avidin coupled to one of the abovelabel groups.

[0169] There are also numerous methods of separating the bound from thefree antigen, or alternatively the bound from the free test compound.The CTLA-8 protein can be immobilized on various matrixes followed bywashing. Suitable matrixes include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the CTLA-8 protein to amatrix include, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin-avidin. The last step inthis approach involves the precipitation of protein-protein complex byany of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magneticparticle separation as described in U.S. Pat. No. 4,659,678.

[0170] The methods for linking proteins or their fragments to thevarious labels have been extensively reported in the literature and donot require detailed discussion here. Many of the techniques involve theuse of activated carboxyl groups either through the use of carbodiimideor active esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

[0171] Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of aCTLA-8 protein. These sequences can be used as probes for detectinglevels of antigen message in samples from patients suspected of havingan abnormal condition, e.g., cancer or developmental problem. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. Normally anoligonucleotide probe should have at least about 14 nucleotides, usuallyat least about 18 nucleotides, and the polynucleotide probes may be upto several kilobases. Various labels may be employed, most commonlyradionuclides, particularly ³²P. However, other techniques may also beemployed, such as using biotin modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed which can recognize specificduplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes,or DNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out in any conventional techniques such as nucleicacid hybridization, plus and minus screening, recombinational probing,hybrid released translation (HRT), and hybrid arrested translation(HART). This also includes amplification techniques such as polymerasechain reaction (PCR).

[0172] Diagnostic kits which also test for the qualitative orquantitative presence of other markers are also contemplated. Diagnosisor prognosis may depend on the combination of multiple indications usedas markers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97.

[0173] The broad scope of this invention is best understood withreference to the following examples, which are not intended to limit theinvention to specific embodiments.

EXAMPLES

[0174] I. General Methods

[0175] Some of the standard methods are described or referenced, e.g.,in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York; Innis, et al.(eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, N.Y. Methods for protein purification include such methods asammonium sulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification” in Methods in Enzymology, vol. 182, and other volumes inthis series; and manufacturer's literature on use of proteinpurification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad,Richmond, Calif. Combination with recombinant techniques allow fusion toappropriate segments, e.g., to a FLAG sequence or an equivalent whichcan be fused via a protease-removable sequence. See, e.g., Hochuli(1989) Chemische Industrie 12:69-70; Hochuli (1990) “Purification ofRecombinant Proteins with Metal Chelate Absorbent” in Setlow (ed.)Genetic Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;and Crowe, et al. (1992) QIAexpress: The High Level Expression & ProteinPurification System QUIAGEN, Inc., Chatsworth, Calif.

[0176] FACS analyses are described in Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; and Robinson, et al.(1993) Handbook of Flow Cytometry Methods Wiley-Liss, New York, N.Y.

[0177] II. Isolation of a DNA Clone Encoding CTLA-8 Protein.

[0178] Isolation of murine CTLA-8 is described in Rouvier, et al. (1993)J. Immunol. 150:5445-5456.

[0179] Source of the CTLA-8 Message

[0180] Various cell lines are screened using an appropriate probe forhigh level message expression. Appropriate cell lines are selected basedupon expression levels of the CTLA-8 message. Applicants usedsubtractive hybridization methods on activated cytotoxic T cells.

[0181] Isolation of a CTLA-8 Encoding Clone

[0182] Standard PCR techniques are used to amplify a CTLA-8 genesequence from a genomic or cDNA library, or from mRNA. Appropriateprimers are selected from the sequences provided, and a full lengthclone is isolated. Various combinations of primers, of various lengthsand possibly with differences in sequence, may be prepared. The fulllength clone can be used as a hybridization probe to screen for otherhomologous genes using stringent or less stringent hybridizationconditions.

[0183] In another method, oligonucleotides are used to screen a library.In combination with polymerase chain reaction (PCR) techniques,synthetic oligonucleotides in appropriate orientations are used asprimers to select correct clones from a library.

[0184] III. Isolation of a Human CTLA-8.

[0185] A human genomic library was obtained from Clontech (Cat. HL1006d)and screened with a cDNA probe composed of a 453 base pair entire codingsequence of a murine CTLA-8. A number of independent lambda clones werefound to hybridize strongly with the murine CTLA-8 probe. One clonecontained a hybridizing XbaI fragment of approximately 2000 base pairswhich corresponded to a fragment previously detected using a similarprobe on a human genomic DNA Southern blot. This 2000 base pair fragmentwas subcloned into Bluescript (Stratagene) and sequenced. This revealeda 240 base pair region (see Table 3) 83.8% homologous to the murineCTLA-8 of Table 1. Translation of this region yielded an amino acidsequence 70.8% homologous to the 79 carboxy-terminal amino acids of themurine CTLA-8 putative protein. The exon was used as a probe to screen alibrary of cDNA made with a primer corresponding to the last 21nucleotides of the coding region. Three independent cDNA clones wereobtained containing the complete coding region of the human CTLA-8. The468 base pair open reading frame encodes a 155 amino acid polypeptidewith a theoretical molecular weight of 17,100 daltons. See Table 3. Thishuman CTLA-8 is 66.4% homologous to the ORF-13 of the virus, and 58.3%homologous to murine CTLA-8 encoded protein. Moreover, the 6 cysteinesare conserved between the three genes, as well as the putativeglycosylation and phosphorylation sites.

[0186] Analysis of the human CTLA-8 amino acid sequence exhibits ahydrophobic stretch of 19 residues, from 7 to about 25, at the aminoterminus, similar to a signal peptide. It is highly likely that thehuman CTLA-8 is a secreted protein of a molecular weight resembling acytokine.

[0187] IV. Biochemical Characterization of CTLA-8 Proteins.

[0188] Two forms of human CTLA-8 were expressed in heterologous cells;the native form, and a recombinant form displaying the FLAG peptide atthe carboxy terminus. See, e.g., Crowe et al. (1992) QIAexpress: TheHigh Level Expression and Protein Purification System QIAGEN, Inc.Chatsworth, Calif.; and Hopp et al. (1988) Bio/Technology 6:1204-1210.These two forms of the human CTLA-8 protein were introduced into theexpression vectors pME18S or pEE12, and subsequently transfected intoCOS-7 or NSO cells, respectively, by electroporation. Electroporatedcells were then cultivated for 48 hours in RPMI medium supplemented with10% Fetal Calf Serum. Cells were then incubated with ³⁵S-Met and ³⁵S-Cysin order to label cellular proteins. Comparison of the proteins underreducing conditions on SDS-PAGE showed that cells transfected with humanCTLA-8 secreted a polypeptide of 15,000 daltons. Non-reducing SDS-PAGErevealed 2 specific bands around 28,000 daltons and 33,000 daltons.Treatment with endoglycosidase F (Boehringer Mannheim) demonstrated thatthe higher molecular weight species represents an N-glycosylated form ofhuman CTLA-8.

[0189] In order to determine if the natural form of human CTLA-8produced by activated CD4+ T cells was also secreted as a dimer similarto transfected COS-7 and NSO cells, peripheral blood mononuclear cells(PBMC) were purified from 500 ml of human blood on a Ficoll gradient. Bcells, CD8+ T cells, monocytes, and NK cells were depleted using 100 μlof ascitic fluid containing anti-CD19, anti-CD8, anti-CD14, and 25 gg ofNKH1 monoclonal antibody (Coulter, Hialeah, Fla.). After 30 minutes ofincubation at 40° C., the PBMC were washed twice in RPMI containing 10%Fetal Calf Serum (FCS). Paramagnetic beads coated with goat antibodiesto mouse IgG (Dynabeads M450, Dynal, Oslo, Norway) were added at a finalconcentration of 5 beads/cell to be depleted. Unwanted cells weresubsequently removed by 3 passages on a magnet. The remaining cells wereCD4+ cells at 87% purity which were diluted to 10⁷ cells/ml in DMEM F12(Gibco, Gaithersburg, Md.) containing 10% FCS, 10 ng/ml PMA (Sigma, St.Louis, Mo.) and 500 ng/ml ionomycin (Sigma, St. Louis, Mo.). Afterincubation for 4 hours at 37° C. in 5% CO₂, the medium was changed tomethionine and cysteine free DMEM (ICN Biomedicals, Costa Mesa, Calif.),supplemented with 1% dialyzed FCS, 10 ng/ml PMA and 500 ng/ml ionomycin,and incubated for 1 hour at 37° C. in 5% CO₂. 100 μCi/ml of³⁵S-methionine and ³⁵S-cysteine (Amersham) was added, and metaboliclabeling was carried out for 18 hours at 37° C. in 5% CO₂. Followingpreclearing of the supernatants with anti-IFN-γ Mab B27 and 0.5 ml ofProtein-G Sepharose (Sigma St. Louis, Mo.), the supernatants wereimmunoprecipated using monoclonal antibodies to human CTLA-8.Immunoprecipated proteins were analyzed on SDS-PAGE. CD4+ T cells andtransfected NSO cells reveal two bands at 28,000 and 33,000 daltonscorresponding respectively to non N-glycosylated and N-glycosylatedforms of human CTLA-8 dimers. Therefore, human CTLA-8 derived fromtransfected NSO cells and CTLA-8. isolated from activated T cellsdisplay the same biological characteristics.

[0190] V. Large Scale Production of Human CTLA-8

[0191] For biological assays, human CTLA-8 and human CTLA-8-FLAG wereproduced in large amounts with transfected COS-7 cells grown in RPMImedium supplemented with 1% Nutridoma HU (Boeringer Mannheim, Mannheim,Germany) and subsequently purified.

[0192] In order to produce larger quantities of native human CTLA-8 orhuman CTLA-8-FLAG, stable transformants of NSO cells were preparedaccording to the methodology developed by Celltech (Slough, Berkshire,UK; International Patent Applications WO86/05807, WO87/04462,WO89/01036, and WO89/10404). Both CTLA-8 and CTLA-8-FLAG were subclonedinto pEE12 and subsequently transfected into NSO cells byelectroporation. Transfected NSO cells were seeded in selectiveglutamine-free DMEM supplemented with 10% Fetal Calf Serum as describedin Celltech's protocol. Supernatants from the best producing lines wereused in biological assays and purification of human CTLA-8 and humanCTLA-8-FLAG.

[0193] Purification of Human CTLA-8 Protein

[0194] Typically, 1 liter of supernatant containing human CTLA-8 orCTLA-8-FLAG was passed on a 60 ml column of Zn⁺⁺ ions grafted to aChelating Sepharose Fast Flow matrix (Pharmacia, Upsalla, Sweden). Afterwashing with 10 volumes of binding buffer (His-Bind Buffer kit, Novagen,Madison, Wis.), the proteins retained by the metal ions were eluted witha gradient of 20-100 mM Imidazole. The content of human CTLA-8-FLAG inthe eluted fractions was determined by dot blot using the anti-FLAGmonoclonal antibody M2 (Eastman Kodak, New-Haven, Conn.), whereas thecontent of human CTLA-8 was assessed by silver staining of non-reducingSDS-PAGE. The CTLA-8 containing fractions were then pooled and dialyzedagainst PBS, and were either used in biological assays or furtherpurified by anion exchange HPLC on a DEAE column. A third step of glefiltration chromatograph was performed on a SUPERDEX G-75 HRD30 column(Pharmacia Uppsala, Sweden) and yielded practically pure human CTLA-8-8as analyzed by silver stained SDS-PAGE.

[0195] Preparation of Antibodies Specific for CTLA-8

[0196] Inbred Balb/c mice were immunized intraperitoneally with 1 ml ofpurified human CTLA-8-FLAG emulsified in Freund's complete adjuvant onday 0, and in Freund's incomplete adjuvant on days 15 and 22. The micewere boosted with 0.5 ml of purified human CTLA-8-8 administeredintravenously.

[0197] Hybridomas were created using the non-secreting myeloma cellsline SP2/0-Ag8 and polyethylene glycol 1000 (Sigma, St. Louis, Mo.) asthe fusing agent. Hybridoma cells were placed in a 96-well Falcon tissueculture plate (Becton Dickinson, N.J.) and fed with DMEM F12 (Gibco,Gaithersburg, Md.) supplemented with 80 μg/ml gentamycin, 2 mMglutamine, 10% horse serum (Gibco, Gaithersburg, Md.), 1% ADCM (CRTS,Lyon, France:) 10⁻⁵ M azaserine (Sigma, St. Louis, Mo.) and 5×10⁻⁵ Mhypoxanthine. Hybridoma supernatants were screened for antibodyproduction against human CTLA-8 by immunocytochemistry (ICC) usingacetone fixed human CTLA-8 transfected COS-7 cells and by ELISA usinghuman CTLA-8-FLAG purified from COS-7 supernatants as a coating antigen.Aliquots of positive cell clones were expanded for 6 days andcryopreserved as well as propagated in ascites from pristane(2,6,10,14-teramethylpentadecane, Sigma, St. Louis, Mo.) treated Balb/cmice who had received on intraperitoneal injection of pristane 15 daysbefore. About 10⁵ hybridoma cells in 1 ml of PBS were givenintraperitoneally, and 10 days later, ascites were collected from eachmouse.

[0198] After centrifugation of the ascites, the antibody fraction wasisolated by ammonium sulfate precipitation and anion-exchangechromatography on a zephyr-D silicium column (IBF Sepracor) equilabratedwith 20 mM Tris pH 8.0. Proteins were eluted with a NaCl gradient(ranging from 0 to 1 M NaCl). 2 ml fractions were collected and testedby ELISA for the presence of anti-CTLA-8 antibody. The fractionscontaining specific anti-CTLA-8 activity were pooled, dialyzed, andfrozen. Aliquots of the purified monoclonal antibodies were peroxydaselabeled.

[0199] Quantification of Human CTLA-8

[0200] Among the antibodies specific for CTLA-8, Ab25, and peroxydaselabeled Ab16 were selected to quantitate levels of human CTLA-8 using asandwich assay. Purified Ab25 was diluted at 2 μg/ml in coating buffer(carbonate buffer, pH 9.6. 15 mM Na₂CO₃, 35 mM NaHCO₃). This dilutedsolution was coated onto the wells of a 96-well ELISA plate (ImmunoplateMaxisorp F96 certified, NUNC, Denmark) overnight at room temperature.The plates were then washed manually one with a washing bufferconsisting of Phosphate Buffered Saline and 0.05% Tween 20 (TechniconDiagnositics, USA). 110 μl of purified human CTLA-8 diluted in TBS-B-Tbuffer [20 mM Tris, 150 mM NaCl, 1% BSA (Sigma, St. Louis, Mo.), and0.05% Tween 20] was added to each well. After 3 hours of incubation at37° C., the plates were washed once. 100 μl of peroxydase labeled Ab16diluted to 5 μg/ml in TBS-B-T buffer was added to each well, andincubated for 2 hours at 37° C. The wells were then washed three timesin washing buffer. 100 μl of peroxydase substrate, 2.2′ Azino-bis(3ethylbenzthiazoine-6-sulfonic acid) (ABTS), diluted to 1 mg/ml incitrate/phosphate buffer, was added to each well, and the colorimetricreaction was read at 405 nm. The lowest concentration of human CTLA-8detected was 0.015 ng/ml.

[0201] V. Induction of IL-6 Secretion by Treatment of Various Cell Typeswith CTLA-8

[0202] Synoviocytes from normal and rheumatoid arthritic patients (10⁴cells/well) were incubated with increasing concentrations of humanCTLA-8-8. After 48 hours, concentrations of IL-6 were measured bystandard ELISA techniques. Secretion of IL-6 was increased in both typesof cells in a dose dependent manner.

[0203] Kidney epithelial carcinoma cell lines TUMT and CHA were alsocultured in complete RPMI 1640 medium (Gibco BRL, Grand Island, N.Y.),supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 μg/mlgentamycin, 20 mM Hepes buffer and heat-inactivated 10% FCS. Cells (104cells/well) were incubated in 96-well plates (Falcon) in a final volumeof 250 μl of complete culture medium. Increasing concentrations of humanCTLA-8-8 were added at the onset of the culture. Cell-free supernatantswere collected after 48 hours, and stored at −20° C. until cytokineassays. IL-6 levels were measured by two-site sandwich ELISA asdescribed in Abrams, et al. (1992). Immunol. Rev. 127:5-24. Both celllines exhibited dose dependent increases in IL-6 secretion withincreasing concentrations of CTLA-8. In view of these results, othercell lines will also be screened for responses to other species ofCTLA-8 variants.

[0204] MRC-5 human lung fibroblasts were obtained from the ATCC(Rockville, Md.) and were cultured in complete RPMI 1640 medium (GibcoBRL, Grand Island, N.Y.), supplemented with 2 mM L-glutamine, 100 U/mlpenicillin, 50 mg/ml gentamycin, 20 mM Hepes buffer and heat-inactivated10% FCS. Cells (10⁴ cells/well) were incubated in 96-well plates(Falcon) in a final volume of 250 ml of complete culture medium.Increasing concentrations of human CTLA-8-8 was added at the onset ofthe culture. Cell-free supernatants were collected after 48 hours, andstored at −20° C. until cytokine assays. IL-6 levels, measured by ELISA.Dose dependent induction of IL-6 was observed.

[0205] Similar results were obtained using adult and child dermalfibroblasts, human brain epithelial cells, and human bronchus epithelialcells. Kidney mesangium cells are also expected to respond similarly.

[0206] VI. Isolating CTLA-8 Homologues

[0207] The binding composition is used for screening of an expressionlibrary made from a cell line which expresses a CTLA-8 protein. Standardstaining techniques are used to detect or sort intracellular or surfaceexpressed antigen, or surface expressing transformed cells are screenedby panning. Screening of intracellular expression is performed byvarious staining or immunofluorescence procedures. See also McMahan, etal. (1991) EMBO J. 10:2821-2832.

[0208] For example, on day 0, precoat 2-chamber permanox slides with 1ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min at roomtemperature. Rinse once with PBS. Then plate COS cells at 2-3×10⁵ cellsper chamber in 1.5 ml of growth media. Incubate overnight at 37° C.

[0209] On day 1 for each sample, prepare 0.5 ml of a solution of 66μg/ml DEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum free DME.For each set, a positive control is prepared, e.g., of huIL-10-FLAG cDNAat 1 and 1/200 dilution, and a negative mock. Rinse cells with serumfree DME. Add the DNA solution and incubate 5 hr at 37° C. Remove themedium and add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and wash oncewith DME. Add 1.5 ml growth medium and incubate overnight.

[0210] On day 2, change the medium. On days 3 or 4, the cells are fixedand stained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80° C. after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin (0.1%) with 32 μl/ml of 1 M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Soluble antibody is added to cells andincubate for 30 min. Wash cells twice with HBSS/saponin. Add secondantibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, andincubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABChorseradish peroxidase solution, and preincubate for 30 min. Use, e.g.,1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 mlHBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solutionand incubate for 30 min. Wash cells twice with HBSS, second wash for 2min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of H₂O₂per 5 ml of glass distilled water. Carefully remove chamber and rinseslide in water. Air dry for a few minutes, then add 1 drop of CrystalMount and a cover slip. Bake for 5 min at 85-90° C.

[0211] Alternatively, the binding compositions are used to affinitypurify or sort out cells expressing the antigen. See, e.g., Sambrook, etal. or Ausubel, et al.

[0212] Similar methods are applicable to isolate either species orallelic variants. Species variants are isolated using cross-specieshybridization techniques based upon a full length isolate or fragmentfrom one species as a probe, or appropriate species.

[0213] All references cited herein are incorporated herein by referenceto the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

[0214] Many modifications and variations of this invention can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

SEQUENCE SUBMISSION

[0215] SEQ ID NO: 1 is murine CTLA-8 cDNA nucleic acid sequence.

[0216] SEQ ID NO: 2 is murine CTLA-8 peptide amino acid sequence.

[0217] SEQ ID NO: 3 is herpesvirus ORF13 nucleic acid sequence.

[0218] SEQ ID NO: 4 is predicted ORF13 amino acid sequence.

[0219] SEQ ID NO: 5 is human CTLA-8 cDNA nucleic acid sequence.

[0220] SEQ ID NO: 6 is predicted human CTLA-8 amino acid sequence.

[0221] SEQ ID NO: 7 is human CTLA-8 cDNA nucleic acid sequence.

[0222] SEQ ID NO: 8 is predicted human CTLA-8 amino acid sequence.

[0223] SEQ ID NO: 9 is mouse CTLA-8 cDNA nucleic acid sequence.

[0224] SEQ ID NO: 10 is mouse CTLA-8 predicted amino acid sequence.

1 10 1080 base pairs nucleic acid single linear cDNA CDS 12..464/product= “mouse/rat CTLA-8” 1 GAATTCCATC C ATG TGC CTG ATG CTG TTG CTGCTA CTG AAC CTG GAG GCT 50 Met Cys Leu Met Leu Leu Leu Leu Leu Asn LeuGlu Ala 1 5 10 ACA GTG AAG GCA GCG GTA CTC ATC CCT CAA AGT TCA GTG TGTCCA AAC 98 Thr Val Lys Ala Ala Val Leu Ile Pro Gln Ser Ser Val Cys ProAsn 15 20 25 GCC GAG GCC AAT AAC TTT CTC CAG AAC GTG AAG GTC AAC CTG AAAGTC 146 Ala Glu Ala Asn Asn Phe Leu Gln Asn Val Lys Val Asn Leu Lys Val30 35 40 45 ATC AAC TCC CTT AGC TCA AAA GCG AGC TCC AGA AGG CCC TCA GACTAC 194 Ile Asn Ser Leu Ser Ser Lys Ala Ser Ser Arg Arg Pro Ser Asp Tyr50 55 60 CTC AAC CGT TCC ACT TCA CCC TGG ACT CTG AGC CGC AAT GAG GAC CCT242 Leu Asn Arg Ser Thr Ser Pro Trp Thr Leu Ser Arg Asn Glu Asp Pro 6570 75 GAT AGA TAT CCT TCT GTG ATC TGG GAG GCA CAG TGC CGC CAC CAG CGC290 Asp Arg Tyr Pro Ser Val Ile Trp Glu Ala Gln Cys Arg His Gln Arg 8085 90 TGT GTC AAC GCT GAG GGG AAG TTG GAC CAC CAC ATG AAT TCT GTT CTC338 Cys Val Asn Ala Glu Gly Lys Leu Asp His His Met Asn Ser Val Leu 95100 105 ATC CAG CAA GAG ATC CTG GTC CTG AAG AGG GAG CCT GAG AAG TGC CCC386 Ile Gln Gln Glu Ile Leu Val Leu Lys Arg Glu Pro Glu Lys Cys Pro 110115 120 125 TTC ACT TTC CGG GTG GAG AAG ATG CTG GTG GGC GTG GGC TGC ACCTGC 434 Phe Thr Phe Arg Val Glu Lys Met Leu Val Gly Val Gly Cys Thr Cys130 135 140 GTT TCC TCT ATT GTC CGC CAT GCG TCC TAAACAGAGA CCTGAGGCTA481 Val Ser Ser Ile Val Arg His Ala Ser 145 150 GCCCCTAAGA AACCCCTGCGTTTCTCTGCA AACTTCCTTG TCTTTTTAAA ACAGTTCACACA 541 GTTGAATCTC AGCAAGTGATATGGATTTAA AGGCGGGGTT AGAATTGTCT GCCTTCCACACC 601 CTGAAAAGAA GGCGCAGAGGGGATATAAAT TGCTTCTTGT TTTTCTGTGG GCTTTAAATATT 661 ATTTATGTAT TTACTCTATCCCGAGATAAC TTTGAGGCAT AAGTTATTTT AATGAATTATAT 721 CTACATTATT ATTATGTTTCTTAATGCAGA AGACAAAATT CAAGACTAAG AAATTTTATATT 781 ATTTAAAAGG TAAAACCTATATTTATATGA GCTATTTATG GGTCTATTTA TTTTTCTTTCAC 841 GTGCTAAGAT CATGATTATCAGATCTACCT AAGGAAGTCC TAAATAATAT TAAATATTATAA 901 TTGAAATTTC AGTTTTACTATTTGCTTATT TAAGGTTCCC TCCTCTGAAT GGTGTGAAAAAT 961 CAAACCTCGT TTTATGTTTTTAAATTATTG AGGCTTCGAA AAATTGGGCA ATTTAGCTCTTC 1021 CTACTGTGTG TTTAAAAACCTTGTAACAAT ATCACTATAA TAAATTTTTG GAAGAAAAAAAT 1080 150 amino acids aminoacid linear protein 2 Met Cys Leu Met Leu Leu Leu Leu Leu Asn Leu GluAla Thr Val Lys 1 5 10 15 Ala Ala Val Leu Ile Pro Gln Ser Ser Val CysPro Asn Ala Glu Ala 20 25 30 Asn Asn Phe Leu Gln Asn Val Lys Val Asn LeuLys Val Ile Asn Ser 35 40 45 Leu Ser Ser Lys Ala Ser Ser Arg Arg Pro SerAsp Tyr Leu Asn Arg 50 55 60 Ser Thr Ser Pro Trp Thr Leu Ser Arg Asn GluAsp Pro Asp Arg Tyr 65 70 75 80 Pro Ser Val Ile Trp Glu Ala Gln Cys ArgHis Gln Arg Cys Val Asn 85 90 95 Ala Glu Gly Lys Leu Asp His His Met AsnSer Val Leu Ile Gln Gln 100 105 110 Glu Ile Leu Val Leu Lys Arg Glu ProGlu Lys Cys Pro Phe Thr Phe 115 120 125 Arg Val Glu Lys Met Leu Val GlyVal Gly Cys Thr Cys Val Ser Ser 130 135 140 Ile Val Arg His Ala Ser 145150 2520 base pairs nucleic acid single linear DNA (genomic) CDS1574..2029 /product= “Saimiriine herpesvirus 2 immediate-early protein”/note= “open reading frame 2 (ORF2)” 3 AGCTTCATGC AAATACATCT TATCTTACCAGATTCTCGCC TCATTTGCAA ACATGCCTCA 60 TCTTTTGAGA AGAAACGCAA TTCGAACTTCTTCTAATGCT CCTGAAGAGC AGCCTGTGCT 120 GCAGCCTGAG CTTGATGCTA TTGAAGAGCTAGAATAAGAG CTATTTTTTG ACGATGGGTG 180 CTGCCTTTCT GTTCAAGAAA TCTGCTTAATTGTTCTTGGA TTCTTATTGT TTCTGCTAGC 240 TGTAATTGTT TTTTATAACT ATACAGACACAGATCAATTT GTGAAGCTGA CACATCTTAT 300 GAGCCACAAA AATTCTATCA AAGGACCTTTTGATCTTTAA GGTATGTACT CATAATTTTA 360 TTTTTTTATT TCTAAAACAA TCTTAGTATATATAATTAAT ACAAATTTTA GAAAATACTA 420 TAATAAATAT TGAAAGCTGT ATTTACATTGTAAACTATAT ATAGGCAATG TAAAGTCATT 480 CTAACTTTAG GTTTGCTTTA CCTGTTACAGAAACTTCACC TGTGTGTCAA GAGCTGCAAA 540 CATGGCTTTA GACTTAAGAA ATCTTAAACACCTGACTGCT AACTTCAGTT TTAGAATAAT 600 GATATGGATT ATGCTATGTT TGGCTCTACCTACTGATAGT AAACCTATTT CAACAACTGA 660 AGCTCCAATA CTAAACATAA CACAATCTCCAAGTTTGAAC ATCTCATCAC CTTCTACTTT 720 AGAACCTTCA GAGCCTCTTA AAAACTGTACAACATTCTTA GACTTACTTT GGCAGCGGCT 780 GGGCGAGAAC GCTTCTATAA AGGACTTGATGTTAACATTA CAACGAGAAG AAGTCCACGG 840 AAGAATGACT ACACTTCCTT CACCTAGACCAAGCAGTAAA GTTGAAGAAC AACAGTTACA 900 AAGACCTAGA AACTTACTGC CTACTGCTGTCGGGCCACCT CATGTCAAAT ATAGACTATA 960 TAATCGCTTA TGGGAAGCTC CTAAAGGAGCTGATGTTAAT GGTAAACCTA TACAATTTGA 1020 TGACCCTCCT CTTCCTTATA CAGGGGCATATAATGATGAT GGTGTTTTAA TGGTTAATAT 1080 TAATGGAAAA CATGTGAGGT TTGATAGCTTGTCTTATTGG GAAAGAATTA AAAGATCTGG 1140 TACCCCATGG TGTATAAAGA CACCAAGTGAAAAAGCAGCA ATATTGAAGC AGCTTTTAAA 1200 AGCTGAAAAA AAATGTAGGA CTACTTCTAAACGTATCACT GAGTTAGAAG AGCAGATTAA 1260 AGAACTAGAA AAAACTAGTA CATCTCCATAGATTACTGTT AGAATGTGTT TATCATACTA 1320 AAATAAATGC TTTATGTATT GCAATATTACTTGTTTGCTA TGACTTTGGT ATATGAAATA 1380 CAAATCTTAA ATAAAAAGTT TTTGTCTAGTATTGGCGTCA CTGTATTTTA CTAGCAAAAA 1440 TATATAAATT GTTATGTAGC AAGAAGTTTGTATCAATATA AAAACTCTAA AGTATATAAA 1500 CAAACATTCA ATTAGTGTAA ATCATAGCAAGCATATCTTT TCATACGTGT CTAGTTAATT 1560 TAAAGAATTA ATT ATG ACA TTT AGA ATGACT TCA CTT GTG TTA CTT CTG 1609 Met Thr Phe Arg Met Thr Ser Leu Val LeuLeu Leu 1 5 10 CTG CTG AGC ATA GAT TGT ATA GTA AAG TCA GAA ATA ACA AGCGCA CAA 1657 Leu Leu Ser Ile Asp Cys Ile Val Lys Ser Glu Ile Thr Ser AlaGln 15 20 25 ACC CCA AGA TGC TTA GCT GCT AAC AAT AGC TTT CCA CGG TCT GTGATG 1705 Thr Pro Arg Cys Leu Ala Ala Asn Asn Ser Phe Pro Arg Ser Val Met30 35 40 GTT ACT TTG AGC ATC CGT AAC TGG AAT ACT AGT TCT AAA AGG GCT TCA1753 Val Thr Leu Ser Ile Arg Asn Trp Asn Thr Ser Ser Lys Arg Ala Ser 4550 55 60 GAC TAC TAC AAT AGA TCT ACG TCT CCT TGG ACT CTC CAT CGC AAT GAA1801 Asp Tyr Tyr Asn Arg Ser Thr Ser Pro Trp Thr Leu His Arg Asn Glu 6570 75 GAT CAA GAT AGA TAT CCC TCT GTG ATT TGG GAA GCA AAG TGT CGC TAC1849 Asp Gln Asp Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys Cys Arg Tyr 8085 90 TTA GGA TGT GTT AAT GCT GAT GGG AAT GTA GAC TAC CAC ATG AAC TCA1897 Leu Gly Cys Val Asn Ala Asp Gly Asn Val Asp Tyr His Met Asn Ser 95100 105 GTC CCT ATC CAA CAA GAG ATT CTA GTG GTG CGC AAA GGG CAT CAA CCC1945 Val Pro Ile Gln Gln Glu Ile Leu Val Val Arg Lys Gly His Gln Pro 110115 120 TGC CCT AAT TCA TTT AGG CTA GAG AAG ATG CTA GTG ACT GTA GGC TGC1993 Cys Pro Asn Ser Phe Arg Leu Glu Lys Met Leu Val Thr Val Gly Cys 125130 135 140 ACA TGC GTT ACT CCC ATT GTT CAC AAT GTA GAC TAAAAGCTATCTAAATTT 2046 Thr Cys Val Thr Pro Ile Val His Asn Val Asp 145 150AAAATTAACA TTTCACTAAA AAACAAAAAC TTGATTTTTT TCTTTTAAAT AAAAAAAGTT 2106TAATATAAGT TCTGGCTTGT TTGGTTTTTG ACTAATCAAT GTAGATCACA CTTGTGATCT 2166TAGCTCTCGG GAAGCAATGT AAGAAAATAT ATTTAACTTA AGAGTTTTAG ACTTGCTTGA 2226GTTTTATGAG TAAAAAACAA AGAATAAGCA CAGCTTCTTG TATCTTCTTT TAAAAACTTT 2286AAGTTATTTA TGTATTTAAT ATAATCTAAT GTTTCTTAAA CATGTTGAGT TTGAGGTCCA 2346CTAATACAAC ATTATAATTT TTTCTGTTAT AACACTTTTG CAAGAAGAAC TCATTTTATA 2406GAAAATGAGC AGTATTCAAA AAAAATGTTT GATATGCTGT AATATTGGAG AGGAAGAACT 2466TTTACAAGCA TGTGATTGTC CTAGCAGAGT CCATCATACA TGCTTACAAA GTCA 2520 151amino acids amino acid linear protein 4 Met Thr Phe Arg Met Thr Ser LeuVal Leu Leu Leu Leu Leu Ser Ile 1 5 10 15 Asp Cys Ile Val Lys Ser GluIle Thr Ser Ala Gln Thr Pro Arg Cys 20 25 30 Leu Ala Ala Asn Asn Ser PhePro Arg Ser Val Met Val Thr Leu Ser 35 40 45 Ile Arg Asn Trp Asn Thr SerSer Lys Arg Ala Ser Asp Tyr Tyr Asn 50 55 60 Arg Ser Thr Ser Pro Trp ThrLeu His Arg Asn Glu Asp Gln Asp Arg 65 70 75 80 Tyr Pro Ser Val Ile TrpGlu Ala Lys Cys Arg Tyr Leu Gly Cys Val 85 90 95 Asn Ala Asp Gly Asn ValAsp Tyr His Met Asn Ser Val Pro Ile Gln 100 105 110 Gln Glu Ile Leu ValVal Arg Lys Gly His Gln Pro Cys Pro Asn Ser 115 120 125 Phe Arg Leu GluLys Met Leu Val Thr Val Gly Cys Thr Cys Val Thr 130 135 140 Pro Ile ValHis Asn Val Asp 145 150 237 base pairs nucleic acid single linear cDNACDS 1..237 /product= “human CTLA-8 fragment” 5 MGC AAT GAG GAC CCT GAGAGA TAT CCC TCT GTG ATC TGG GAG GCA AAG 48 Xaa Asn Glu Asp Pro Glu ArgTyr Pro Ser Val Ile Trp Glu Ala Lys 1 5 10 15 TGC CGC CAC TTG GGC TGCATC AAC GCT GAT GGG AAC GTG GAC TAC CAC 96 Cys Arg His Leu Gly Cys IleAsn Ala Asp Gly Asn Val Asp Tyr His 20 25 30 ATG AAC TCT GTC CCC ATC CAGCAA GAG ATC CTG GTC CTG CGC AGG GAG 144 Met Asn Ser Val Pro Ile Gln GlnGlu Ile Leu Val Leu Arg Arg Glu 35 40 45 CCT CCA CAC TGC CCC AAC TCC TTCCGG CTG GAG AAG ATA CTG GTG TCC 192 Pro Pro His Cys Pro Asn Ser Phe ArgLeu Glu Lys Ile Leu Val Ser 50 55 60 GTG GGC TGC ACC TGT GTC ACC CCG ATTGTC CAC CAT GTG GCC 234 Val Gly Cys Thr Cys Val Thr Pro Ile Val His HisVal Ala 65 70 75 TAA 237 78 amino acids amino acid linear proteinModified-site 1 /product= “OTHER” /note= “Xaa = Ser or Arg” 6 Xaa AsnGlu Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala Lys 1 5 10 15 CysArg His Leu Gly Cys Ile Asn Ala Asp Gly Asn Val Asp Tyr His 20 25 30 MetAsn Ser Val Pro Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu 35 40 45 ProPro His Cys Pro Asn Ser Phe Arg Leu Glu Lys Ile Leu Val Ser 50 55 60 ValGly Cys Thr Cys Val Thr Pro Ile Val His His Val Ala 65 70 75 510 basepairs nucleic acid single linear cDNA - 1..510 /note= “full length humanCTLA-8 clone” CDS 43..510 /product= “human CTLA-8” 7 GGCACAAACTCATCCATCCC CAGTTGATTG GAAGAAACAA CG ATG ACT CCT GGG 54 Met Thr Pro Gly 1AAG ACC TCA TTG GTG TCA CTG CTA CTG CTG CTG AGC CTG GAG GCC ATA 102 LysThr Ser Leu Val Ser Leu Leu Leu Leu Leu Ser Leu Glu Ala Ile 5 10 15 20GTG AAG GCA GGA ATC ACA ATC CCA CGA AAT CCA GGA TGC CCA AAT TCT 150 ValLys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly Cys Pro Asn Ser 25 30 35 GAGGAC AAG AAC TTC CCC CGG ACT GTG ATG GTC AAC CTG AAC ATC CAT 198 Glu AspLys Asn Phe Pro Arg Thr Val Met Val Asn Leu Asn Ile His 40 45 50 AAC CGGAAT ACC AAT ACC AAT CCC AAA AGG TCC TCA GAT TAC TAC AAC 246 Asn Arg AsnThr Asn Thr Asn Pro Lys Arg Ser Ser Asp Tyr Tyr Asn 55 60 65 CGA TCC ACCTCA CCT TGG AAT CTC CAC CGC AAT GAG GAC CCT GAG AGA 294 Arg Ser Thr SerPro Trp Asn Leu His Arg Asn Glu Asp Pro Glu Arg 70 75 80 TAT CCC TCT GTGATC TGG GAG GCA AAG TGC CGC CAC TTG GGC TGC ATC 342 Tyr Pro Ser Val IleTrp Glu Ala Lys Cys Arg His Leu Gly Cys Ile 85 90 95 100 AAC GCT GAT GGGAAC GTG GAC TAC CAC ATG AAC TCT GTC CCC ATC CAG 390 Asn Ala Asp Gly AsnVal Asp Tyr His Met Asn Ser Val Pro Ile Gln 105 110 115 CAA GAG ATC CTGGTC CTG CGC AGG GAG CCT CCA CAC TGC CCC AAC TCC 438 Gln Glu Ile Leu ValLeu Arg Arg Glu Pro Pro His Cys Pro Asn Ser 120 125 130 TTC CGG CTG GAGAAG ATA CTG GTG TCC GTG GGC TGC ACC TGT GTC ACC 486 Phe Arg Leu Glu LysIle Leu Val Ser Val Gly Cys Thr Cys Val Thr 135 140 145 CCG ATT GTC CACCAT GTG GCC TAA 510 Pro Ile Val His His Val Ala 150 155 155 amino acidsamino acid linear protein 8 Met Thr Pro Gly Lys Thr Ser Leu Val Ser LeuLeu Leu Leu Leu Ser 1 5 10 15 Leu Glu Ala Ile Val Lys Ala Gly Ile ThrIle Pro Arg Asn Pro Gly 20 25 30 Cys Pro Asn Ser Glu Asp Lys Asn Phe ProArg Thr Val Met Val Asn 35 40 45 Leu Asn Ile His Asn Arg Asn Thr Asn ThrAsn Pro Lys Arg Ser Ser 50 55 60 Asp Tyr Tyr Asn Arg Ser Thr Ser Pro TrpAsn Leu His Arg Asn Glu 65 70 75 80 Asp Pro Glu Arg Tyr Pro Ser Val IleTrp Glu Ala Lys Cys Arg His 85 90 95 Leu Gly Cys Ile Asn Ala Asp Gly AsnVal Asp Tyr His Met Asn Ser 100 105 110 Val Pro Ile Gln Gln Glu Ile LeuVal Leu Arg Arg Glu Pro Pro His 115 120 125 Cys Pro Asn Ser Phe Arg LeuGlu Lys Ile Leu Val Ser Val Gly Cys 130 135 140 Thr Cys Val Thr Pro IleVal His His Val Ala 145 150 155 1134 base pairs nucleic acid singlelinear cDNA - 1..1134 /note= “mouse CTLA-8 fragment” CDS 45..521/product= “mouse CTLA-8” 9 GAGGCTCAAG TGCACCCAGC ACCAGCTGAT CAGGACGCGCAAAC ATG AGT CCA GGG 56 Met Ser Pro Gly 1 AGA GCT TCA TCT GTG TCT CTGATG CTG TTG CTG CTG CTG AGC CTG GCG 104 Arg Ala Ser Ser Val Ser Leu MetLeu Leu Leu Leu Leu Ser Leu Ala 5 10 15 20 GCT ACA GTG AAG GCA GCA GCGATC ATC CCT CAA AGC TCA GCG TGT CCA 152 Ala Thr Val Lys Ala Ala Ala IleIle Pro Gln Ser Ser Ala Cys Pro 25 30 35 AAC ACT GAG GCC AAG GAC TTC CTCCAG AAT GTG AAG GTC AAC CTC AAA 200 Asn Thr Glu Ala Lys Asp Phe Leu GlnAsn Val Lys Val Asn Leu Lys 40 45 50 GTC TTT AAC TCC CTT GGC GCA AAA GTGAGC TCC AGA AGG CCC TCA GAC 248 Val Phe Asn Ser Leu Gly Ala Lys Val SerSer Arg Arg Pro Ser Asp 55 60 65 TAC CTC AAC CGT TCC ACG TCA CCC TGG ACTCTC CAC CGC AAT GAA GAC 296 Tyr Leu Asn Arg Ser Thr Ser Pro Trp Thr LeuHis Arg Asn Glu Asp 70 75 80 CCT GAT AGA TAT CCC TCT GTG ATC TGG GAA GCTCAG TGC CGC CAC CAG 344 Pro Asp Arg Tyr Pro Ser Val Ile Trp Glu Ala GlnCys Arg His Gln 85 90 95 100 CGC TGT GTC AAT GCG GAG GGA AAG CTG GAC CACCAC ATG AAT TCT GTT 392 Arg Cys Val Asn Ala Glu Gly Lys Leu Asp His HisMet Asn Ser Val 105 110 115 CTC ATC CAG CAA GAG ATC CTG GTC CTG AAG AGGGAG CCT GAG AGC TGC 440 Leu Ile Gln Gln Glu Ile Leu Val Leu Lys Arg GluPro Glu Ser Cys 120 125 130 CCC TTC ACT TTC AGG GTC GAG AAG ATG CTG GTGGGT GTG GGC TGC ACC 488 Pro Phe Thr Phe Arg Val Glu Lys Met Leu Val GlyVal Gly Cys Thr 135 140 145 TGC GTG GCC TCG ATT GTC CGC CAG GCA GCCTAAACAGAGA CCCGCGGCTG 538 Cys Val Ala Ser Ile Val Arg Gln Ala Ala 150155 ACCCCTAAGA AACCCCCACG TTTCTCAGCA AACTTACTTG CATTTTTAAA ACAGTTCGTC598 CTATTGATTT TCAGCAAGGA ATGTGGATTC AGAGGCAGAT TCAGAATTGT CTGCCCTCCA658 CAATGAAAAG AAGGTGTAAA GGGGTCCCAA ACTGCTTCGT GTTTGTTTTT CTGTGGACTT718 TAAATTATTT GTGTATTTAC AATATCCCAA GATAACTTTG AAGGCGTAAC TTATTTAATG778 AAGTATCTAC ATTATTATTA TGTTTCTTTC TGAAGAAGAC AAAATTCAAG ACTCAGAAAT838 TTTATTATTT AAAAGGTAAG CCTATATTTA TATGAGCTAT TTATGAATCT ATTTATTTTT898 CTTCAGTATT TGAAGTATTA AGAACATGAT TTTCAGATCT ACCTAGGGAA GTCCTAAGTA958 AGATTAAATA TTAATGGAAA TTTCAGCTTT ACTATTTGGT TGATTTAAGG TTCTCTCCTC1018 TGAATGGGGT GAAAACCAAA CTTAGTTTTA TGTTTAATAA CTTTTTAAAT TATTGAAGT1078 TCAAAAAATT GGATAATTTA GCTCCCTACT CTGTTTTAAA AAAAAAAAAA AAAAAA 1134158 amino acids amino acid linear protein 10 Met Ser Pro Gly Arg Ala SerSer Val Ser Leu Met Leu Leu Leu Leu 1 5 10 15 Leu Ser Leu Ala Ala ThrVal Lys Ala Ala Ala Ile Ile Pro Gln Ser 20 25 30 Ser Ala Cys Pro Asn ThrGlu Ala Lys Asp Phe Leu Gln Asn Val Lys 35 40 45 Val Asn Leu Lys Val PheAsn Ser Leu Gly Ala Lys Val Ser Ser Arg 50 55 60 Arg Pro Ser Asp Tyr LeuAsn Arg Ser Thr Ser Pro Trp Thr Leu His 65 70 75 80 Arg Asn Glu Asp ProAsp Arg Tyr Pro Ser Val Ile Trp Glu Ala Gln 85 90 95 Cys Arg His Gln ArgCys Val Asn Ala Glu Gly Lys Leu Asp His His 100 105 110 Met Asn Ser ValLeu Ile Gln Gln Glu Ile Leu Val Leu Lys Arg Glu 115 120 125 Pro Glu SerCys Pro Phe Thr Phe Arg Val Glu Lys Met Leu Val Gly 130 135 140 Val GlyCys Thr Cys Val Ala Ser Ile Val Arg Gln Ala Ala 145 150 155

What is claimed is:
 1. A nucleic acid at least 95% identical to oneencoding a mammalian CTLA protein or fragment thereof.
 2. The nucleicacid of claim 1, wherein said encoding nucleic acid comprises a sequenceof SEQ ID NO: 1, 3, 5, 7, or
 9. 3. A substantially pure primate CTLA-8protein or peptide thereof.
 4. The protein or peptide of claim 3,wherein said protein or peptide comprises a sequence of SEQ ID NO: 2, 4,6, 8, or
 10. 5. The protein or peptide of claim 4, wherein said proteinor peptide induces a cell to secrete an inflammatory mediator.
 6. Theprotein or peptide of claim 5, wherein said inflammatory mediator isIL-6; IL-8; and/or PGE2.
 7. A composition comprising a protein of claim3, and a pharmaceutically acceptable carrier.
 8. An antibody whichspecifically binds a mammalian CTLA-8 protein or peptide thereof.
 9. Theantibody of claim 8, wherein said antibody is raised against a peptidesequence of SEQ ID NO: 2, 4, 6, 8, or
 10. 10. The antibody of claim 9,wherein said antibody is a monoclonal antibody.
 11. The antibody ofclaim 10, wherein said antibody blocks a CTLA-8 induced secretion of aninflammatory mediator.
 12. The antibody of claim 10, wherein saidinflammatory mediator is IL-6; IL-8; and/or PGE2.
 13. An antibody ofclaim 10, wherein said antibody is labeled.
 14. A kit comprising: a) anucleic acid at least 95% identical to one encoding a mammalian CTLA-8protein or peptide; b) a substantially pure mammalian CTLA-8 protein orfragment; or c) an antibody or receptor which specifically binds amammalian CTLA-8 protein.
 15. The kit of claim 14, wherein said encodingnucleic acid comprises a sequence of SEQ ID NO: 1, 3, 5, 7, or
 9. 16.The kit of claim 14, wherein said protein or fragment is selected fromthe group consisting of: a) a protein or peptide from a mammal,including a rat or human; b) a protein or peptide comprising at leastone polypeptide segment of SEQ ID NO: 2, 4, 6, 8, or 10; and c) aprotein or peptide which exhibits a post-translational modificationpattern distinct from a natural mammalian CTLA-8 protein.
 17. The kit ofclaim 14 comprising an antibody or receptor, wherein: a) said CTLA-8protein from a mammal, including a mouse or human; b) said antibody israised against a peptide sequence of SEQ ID NO: 2, 4, 6, 8, or 10; c)said antibody is a monoclonal antibody; or d) said antibody is labeled.18. A method of modulating physiology or development of a cellcomprising contacting said cell with an agonist or antagonist of amammalian CTLA-8 protein.
 19. A method of claim 18, wherein saidantagonist is an antibody against a mammalian CTLA-8 protein.
 20. Amethod of modulating CTLA-8 induced secretion of an inflammatorymediator from a cell in a tissue comprising the step of contacting saidtissue with: a) an antibody which specifically binds to a mammalianCTLA-8; or b) a substantially pure CTLA-8 protein or peptide thereof.21. The method of claim 20, wherein said inflammatory mediator isselected from the group consisting of: IL-6; IL-8; and/or PGE2.
 22. Themethod of claim 20, wherein said cell is selected from the groupconsisting of: a) a synovial cell; b) an endothelial cell; c) anepithelial cell; d) a fibroblast cell; or e) a carcinoma cell.